1-[2&#39;,3&#39; -Dideoxy-3&#39; C-(Hydroxymethyl) - Beta-D-Erythro-Pentofuranosyl] Cytosine Derivatives as Hiv

ABSTRACT

Compounds of the formula (I) wherein: R 1  is independently H 1 —OR 3 , —NHR 4 ; C 1 -C 4  alkyl; or, when n is 2, adjacent R 1  together define an olefinic bond; R 2  is H; or when the gem R 1  is C 1 -C 4  alkyl, that R 2  may also be C 1 -C 4  alkyl; or when the gem R 1  is —OR 3 , that R 2  may also be —C(═O)OH or a pharmaceutically acceptable ester thereof; R 3  is independently H, or a pharmaceutically acceptable ester thereof; R 4  is independently H or a pharmaceutically acceptable amide thereof; R 5  and R6 are H or an amine prodrug moiety n is 1, 2 or 3; and pharmaceutically acceptable salts thereof; have utility in the treatment or prophylaxis of HIV, especially reverse transcriptase mutants which allow an obligate chain terminating nucleoside- or nucleotide phosphate to be excised from the nascent DNA strand by ATP- or pyrophosphate-mediated excision.

TECHNICAL FIELD

This invention relates to novel bicyclic tetrahydrofuran derivatives andtheir use in the treatment of retroviruses such as HIV, especially drugescape mutations.

BACKGROUND ART

Unlike other HIV antivirals, such as protease inhibitors ornon-nucleoside reverse transcriptase inhibitors, nucleoside reversetranscriptase inhibitors (NRTI) are pharmacologically inactive in theiradministered form and require phosphorylation by host cellular kinasesto produce the active triphosphate metabolite. This triphosphate formresembles the naturally occurring deoxynucleotide triphosphatesubstrates of the viral reverse transcriptase and competes for HIV-1 RTbinding and incorporation into viral DNA.

All NRTI s approved for the treatment of HIV, and the vast majority ofall other NRTIs proposed in the patent or academic literature, lack a3′-hydroxy function on the ribose moiety of the nucleoside. Examplesinclude zidovudine (AZT), stavudine (d4T), lamivudine (3TC), zalcitabine(ddC), abacavir (ABC), didanosine (ddI) and tenofovir (TNF) (the latterbeing typically administered as the disoproxil fumarate prodrug). Uponphosphorylation, such a nucleoside or nucleotide analogue is covalentlybonded by the reverse transcriptase enzyme to the nascent DNA strand,but the lack of a 3′-hydroxyl function in the nucleoside or nucleotideprevents further attachment of additional nucleotides. These NRTIstherefore terminate viral DNA strand prolongation, thereby leading toinhibition of HIV replication (Mitsuya et al 1990, Jacob Molina et al1993, Reardon 1993).

The cornerstone of all current antiretroviral therapies (ART) is the useof NRTIs. NRTIs, however, are only able to retard HIV propagation in theblood stream and to date have been unable to eradicate HIV frompatients. HIV operates by inserting its DNA into latent host cellsinvolved in human immunologic memory.

This mode of infection implies that patients are forced to take HIVantivirals lifelong in order to prevent the HIV titre from bouncing backafter therapy has ended.

In practice, however, the effective administration period of aparticular HIV drug for a given patient is dramatically limited by theemergence of “escape mutants.” An escape mutant is a virus that containsa discrete cluster of mutations that produces drug resistance and allowsit to proliferate in the presence of the drug. Escape mutants arise in apatient due to the selective pressure of the particular antiviral(s)that the patient is taking. As a consequence, a drug's effectiveadministration period is dependent on how quickly escape mutants ariseand proliferate.

In countries consistently prescribing HIV antivirals it is becomingincreasingly evident that the primary infection in new cases of HIV isoften not with wild type HIV, but rather with a strain of HIV which isalready partly or multiply resistant to the current antivirals. In otherwords, escape mutants which are generated in situ in infected patientscan also be spread to naive patients by lateral or verticaltransmission. This in turn means that even some patients who wouldotherwise be classified as treatment-naive are already infected withvirus resistant to conventional first line therapies.

Multiple factors contribute to the selection of drug escape mutantsincluding total HIV pool size, RT processivity and infidelity in viralgenomic replication, viral fitness and multiple availabilities of targetcells. By the late 1990s, evidence from long term use of combinationsbased on zidovudine (AZT) or stavudine (d4T) suggested that clusters ofparticular mutations in the RT were consistently generated. Thesemutation clusters are the prototype now known as Thymidine AnalogueMutations (TAMs). The presence of TAMs enhanced the likelihood ofselecting further mutations and led to the development of more advancedNRTI resistance phenotypes that were not clearly within the family ofthymidine analogues. Such phenotypes are now known as NucleosideAnalogue Mutation (NAM) and Multiple Drug Resistance (MDR) HIV.

Hypothesis for NRTI Resistance

AZT was the first antiretroviral to be widely used and not surprisinglywas the first to generate escape mutants (Larder et al., 1989). Howeverin view of the large number of mutations throughout the HIV genome intypical patient isolates it is not possible to produce the resistancephenotype in vitro using a recombinant RT enzyme bearing the particularTAM. As a consequence, the mechanisms through which TAMs conferresistance have not been straightforward to elucidate. Varioushypothetical models and theoretical predictions for the mechanism behindTAM resistance have been predicated on the involvement of nucleophilicattack by a pyrophosphate donor (Boyer et al, 2002 and Meyer et al,2002). Presumably RT translocation theory is a key step in understandingthe TAM associated resistance mechanism. This was, however, poorlyunderstood until the end of 2002 because the RT pre- andpost-translocation intermediates are transient and short-lived and notreadily accessed experimentally. The modern understanding of RTtranslocation theory holds that RT catalyzed DNA polymerization takesplace in a detailed cascade fashion as illustrated in FIG. 3, which isadopted from Sarafianos et al (2003). These steps are

-   -   1) Binding of the DNA substrate by free enzyme E positions the        3′-primer end at the P-site (Primer site).    -   2) Binding of a dNTP close to the N-site (dNTP site) forms an        “open” ternary complex.    -   3) A “closed” ternary complex is formed by enzyme conformational        changes.    -   4) Phosphodiester bond formation between the 3′-OH primer        terminus and the alpha phosphate of the dNTP is accompanied by        release of pyrophosphate (PPi) to form the pre-translocated RT        complex at the N-site.    -   5) Translocation of the primer terminus from the N-site to the        P-site by forming a post-translocated complex which is a        prerequisite for the next dNTP binding and continuation of DNA        synthesis.

If a DNA chain terminator nucleoside (NRTI) triphosphate (typically anucleoside analogue which lacks a 3′-hydroxy function on the deoxyribosemoiety) is used, it mimics its natural dNTP counterpart and binds to RT.After the analogous chemical processing, the incorporated NRTI forms apre-translocation complex at the N-site of polymerization. Thisterminates further DNA synthesis due to the lack of a 3′-hydroxyl primeron the NRTI's deoxyribose moiety.

In contrast, TAM-related RT mutations employ a different nucleotideincorporation mechanism compared to wild type RT. Specifically, the newmechanism results in the release (excision) of the NRTI incorporated atthe primer terminus, abrogating the chain terminating activity of theNRTI. This new mechanism is dependent on the interplay between theaccumulation of complexes in pre-translocated states (at the N-site) andthe availability of ATP or pyrophosphate donors, which are oftenabundant at the site of infection, i.e. normal lymphocytes.

ATP or pyrophosphate does not normally participate in viralDNA-polymerization reactions, but the structure of a RT expressing aTAM-related resistant phenotype facilitates their entry into a siteadjacent to a newly incorporated NRTI. The equilibrium between pre- andpost-translocational kinetic species provides a mechanism to ensure freeaccess of the primer terminus to the N-site and also allows simultaneousbinding of the pyrophosphate donor ATP at the P-site after theincorporation of the NRTI chain terminator and the release ofpyrophosphate. When this occurs, ATP (or pyrophosphate) attacks thephosphodiester bond which links the incorporated NRTI at the end of theDNA, resulting in removal of the NRTI via pyrophosphorolysis. When thepyrophosphate donor is ATP, the NRTI is released as a dinucleosidetetraphosphate product. FIG. 4 illustrates this “primer rescue” in anAZT-terminated DNA (adopted from ClinicCareOptions™).

It is now believed that two distinctive mechanisms are involved in thephenotypic resistance to NRTI (Sluis-Cremer et al, 2000). The first,known as “primer rescue” activity, is described immediately above. Here,the chain-terminating nucleotide is removed from the 3′ end of theprimer terminus through ATP-dependent or pyrophosphate-dependentpyrophosphorolysis. There is, however, another cluster of resistancephenotypes denoted as “discriminative mutants.” These mutants have an RTwith enhanced ability to discriminate between NRTIs and native dNTPs. Inthis case, the mechanism leads to RT which is able to preferentiallychoose the right substrate (i.e. native dNTP), thereby avoiding chaintermination by an NRTI and ensuring the propagation of the viral genome.

Generation of Mutations in HIV

Retroviruses such as HIV have the potential for rapid geneticdiversification. While this is an energetically inefficient process, itoffers clear adaptive advantages to the organism. The replicationmachinery used by HIV is particularly error prone, generates a largenumber of mutations and has the potential to lead to accumulation ofmutations when the organism is under selective pressure.

Generally, the vast majority of mutations generated by viral replicationresult in less viable enzymes. Here, the accumulation of a second andespecially a third mutation is less probable because the population poolfor the less viable mutant, within which the second mutation mustaccumulate, will be diluted by the faster multiplying wild typeorganism.

Yet more viable viral mutants can arise and expand by two possiblepathways. The first occurs when there is rapid outgrowth of a highlyresistant variant that is already present in the overall viralpopulation. Most frequently this is a single point mutation that confersphenotypic resistance to a selective pressure. In the context of drugescape mutations examples include K103 rapidly induced by thenon-nucleoside reverse transcriptase inhibitor nevirapine.

The second pathway occurs when there is continued viral replication inthe presence of selective pressure. This allows the progressiveaccumulation of mutations that can then be expanded. In this case, theprobability of mutation accumulation is related to the amount of virusreplication that is occurring. That is, at higher viral loads(e.g. >200,000 copies/ml), accumulations of double mutations can occur.Accumulation of triple mutations, however, are rare and can only resultas a consequence of a complex therapeutic regimen, typically involvingseveral different drugs, that is challenging for the patient to adhereto. It is therefore extremely difficult for even a diligent patient toensure that all active ingredients are present in the blood at levelsabove the necessary inhibitory concentrations over the full 24 hourperiod of each day “24 hour trough level”. Here, temporary removal ofany one of the selective pressures of drug treatment due to lapses inthe administration/24 hour trough level of one or more drugs allowsunbridled viral replication, thereby permitting the generation andestablishment of many new mutants. When the selective pressure is onceagain applied (i.e. resumption of complex drug therapy), the few newmutants that have accumulated another point mutation which confersbetter drug resistance can expand in a manner similar to that seen forthe first pathway (see above).

The discussion above focuses on accumulation of point mutations asopposed to, for example, deletion or addition mutations. Here, however,a scenario similar to that described for a triple mutation isapplicable. That is, most deletion/addition mutations initially involvea single nucleotide. This has the effect of completely altering thedownstream amino acid sequence of the encoded protein if the changeoccurs within the coding region and leads to a truncated and/or inactiveprotein. In order to preserve the reading frame and to alter the finalprotein by the deletion or addition of one single amino acid, threenucleotides must be deleted/added. Since inactive enzymes reduce theviability of an HIV organism, particularly if the enzyme affected is RT,the deletion/additions will not accumulate per se, but must occursimultaneously. In other words the equivalent of a triple mutation mustoccur in a single event, which is highly uncommon (see Boyer et al(2004) J Virol 78(18):9987-9997, which is hereby incorporated byreference in its entirety).

As a consequence of this process for triple mutantaccumulation/introduction, it was not until relatively recently that HIVvirus exhibiting at least three mutations in RT that createsparticularly potent resistance to multiple drugs became established. Forexample, in the United States it was 1992 when the FDA approved the useof combination drug therapy (ddC and AZT). Yet it was not untilSeptember of 1995 that clinical trials showed that the combination ofAZT with ddC or ddI was more effective than AZT alone. It has only beenas a result of the use of combination therapies, where multiple drugsare employed, but in dosage regimes effectively unable to guarantee anadequate 24 hour trough level of the respective drugs, that theparticularly problematic strains of multiresistant H IV virus known inthe Western world today have been generated.

Primer Rescue Mutations

The TAM primer rescue mutant originally described comprised variouspermutations within a group of six drug resistant phenotypes at aminoacid positions M41L, D67N, K70R, L210W, T215Y/F and K219Q/E on RT(Larder and Kemp, 1989, Schinazi et al, 2000). Early data pointed to twodistinctive mutational pathways for the development of multiple TAMprimer rescue mutants, both occurring by unknown factors. The firstpathway resulted in an amino acid substitution at codon 210 (210W) andwas preferentially associated with mutations at codons 41 (41 L; greaterthan 98%) and 215 (215Y; greater than 94%) as well as a substitution atcodon 67 (67N). The second pathway generated a mutation at codon 219(219K/E), which was preferentially associated with mutations at codons67 (67N) and 70 (70R)(Yahi et al, 1999). There were therefore twophenotypic patterns: (1) L210W, M41 L, T215Y/F, ±D67N, which conferredhigh levels of viral resistance to AZT and d4T and (2) K219K/E, D67N,K70R, which conferred moderate levels of viral resistance to AZT andd4T.

Marcelin et al (2004) summarized the prevalence of TAM primerrescue-related mutations in virologic failure pateints. Here, 1098 RTsequences were investigated and gave two genotypic patterns as indicatedin FIG. 1 and FIG. 2. While different genetic backgrounds may have beenpresent prior to therapy, the sequence and composition of theantiretroviral therapy undertaken when combined with individualdifferences in pharmacology resulted in viral resistance not only to AZTand d4T but also to other NRTIs. Depending on the mutational patternpresent, drug resistance included abacavir (ABC), didanosine (ddI),tenofovir (TNF), lamivudine (3TC), emtricitabine (FTC) and zalcitabine(ddC). Hence, the emergence of primer rescue-related TAMs often plays animportant role in the further development of more pronouncedly resistantHIV genotypic patterns. Therefore, one step in preventing multiplenucleoside resistance is to develop a new NRTI with the goal of avoidingthe accumulation of primer rescue related TAMs.

Primer rescue-related TAM mutations can evolve concomitantly with otherfamilies of escape mutants that typically emerge from combinationantiretroviral therapy (otherwise known as cocktail therapy). Today, thecocktail “combivir” (AZT+3TC) is the most frequently used andrecommended first line therapy regimen for treatment of naïve HIVpatients. It leads, however, to escape mutants which are resistant toboth drugs. For example, Miller et al (1998) reported that 3TC-resistantvirus with an M184V mutation was selected just 4-12 weeks afterinitiation of AZT+3TC combination therapy. In time, additionalAZT-associated mutations gradually emerged, giving a characteristicgenotypic pattern of M184V, M41L, D67N, K70R, L210W, T215Y/F and K219Q/Ewhich is commonly found in treatment experienced patients today.Additional mutations in RT at positions H208, R211, and L214 (Sturmer etal, 2003) and at position G333 (Kemp et al 1998) are reported to beinvolved in AZT-3TC double resistance and, in particular, to confer anincrease in the ability to resist AZT. Therefore, the genotypic contextof primer rescue related TAMs has been expanded to include permutationswithin M184V, M41L, D67N, K70R, H208Y, L210W, R211K, L214F, T215Y/F,K219Q/E and G333E.

Other types of mutations generally seen in treatment experiencedpatients are V118I and E44D/A. These mutations are strongly correlatedto prior exposure to ddI and d4T. In addition, they are often associatedwith the presence of specific TAM clusters including M41L plus T215Y/For D67N plus L210W. The result is increased primer rescue-related TAMresistance to the family of thymidine analogues as well as a distinctiverole in the dual resistant to AZT+3TC (Montes et al, 2002, Girouard etal, 2003).

The prevalence of drug escape mutants increases as a function of thenumber of NRTIs used during the course of therapy and forms a pattern ofexpanded TAMs or NAMs comprising various permutations within M41L,E44D/A, D67N, K70R, V118I, M184V, H208Y, L210W, R211K, L214F, T215Y/F,K219Q/E and G333E. This cluster is also commonly refractory to AZT- andd4T-containing combination therapies and cross-resistant to the entireclass of NRTIs.

Significant resistance to thymidine analogues, notably AZT, d4T and TNF,is also found in escape mutants having an amino acid deletion atposition 67(▴67) in the finger region of RT often in association with anamino acid substitution at T69G concomitant with TAM (see Imamichi et al2000 and 2001). An enhanced RT polymerization activity, which isassociated with this particular genotype, is proposed to result in moreefficient pyrophosphorolysis-dependent primer excision (describedabove), leading to the increased resistance Boyer et al, (2004) havealso observed that ▴67 concomitant with TAM conferred an increasedability to facilitate primer rescue (excision) viral resistance to AZTand to TNF as compared to TAM alone.

HIV is co-evolving as antiretroviral therapy develops. New mutationphenotypes emerged when double- and triple-nucleoside analogue cocktailswere employed in the clinical management of HIV, especially intreatment-naive patients. Complex therapeutic regimens, requiringmultiple drugs taken at various times during the day, some with and somewithout food, are challenging for patients. Failure to comply exactlywith these dosing regimes leading to 24 hour trough failures havefacilitated the emergence of multiple NRTI resistant HIV viruses,predominantly as a result of virus acquired NAMs or MDRs. For example, anumber of groups (e.g. Mas et al, 2000) have observed the emergence ofthe mutant T69S-XX virus associated with AZT use. This mutant, has a6-bp insertion in the coding region of its RT between the nucleic acidsspecifying amino acids 69 and 70. The resulting double amino acidinsertion complexes (typically SS, SG or AG insertions) not only led toviral resistance to AZT but also to nearly the entire collection ofNRTIs including d4T, 3TC, ddI, ddC and ABC, and TNF. An enhancedpyrophosphorolysis-dependent primer rescue is seen with the T69S+doubleamino acid insertion, particularly in the presence of TAMs. Thisphenomenon is typically associated with the “M41L/T215Y” or“M41L/L210W/R211K/L214F/T215Y” resistant phenotypes and plays animportant phenotypic role in multiple nucleoside resistance (Meyer etal, 2003).

Another class of MDR has an amino acid substitution at codon Q151M. Thismutation is observed at a relatively low frequency in the clinic andoften presents together with secondary mutations of A62V, V75I, F77L andF116Y. It confers, however, a significant resistance to nearly theentire class of NRTIs. In addition, it has been observed associated withTAMs, typically the “M41L, L210W and T215Y/F” or “D67N, K70R andK219K/E” genotypes. It emerges in patients that have experienced heavytreatment with AZT/ddI and AZT/ddC combination regimens.

L74V is most frequently selected by ddI monotherapy (Martin et al, 1993)and displays cross-resistance to ABC and 3TC. Its effect on producingviral escapes is dependent upon the presence of other mutations.Resistance surveys suggest that the frequency of L74V is linkedsignificantly with TAM, typically in an M41L, L210W and T215Y/Fbackground (Marcelin et al, 2004) even though the L74V mutation wasthought to cause a diminution effect in viral replication and toresensitize AZT-resistant viruses that contain a number of TAMs (St.Clair et al, 1991). A combination of the L74V and M184V mutations inHIV-1 RT is the most frequent pattern associated with resistance to bothABC and ddI (Harrigan et al, 2000 and Miller et al, 2000).

Although high-level resistance to ABC typically requires multiplemutations comprising K65R, L74V, Y115F and M184V, a single mutation,M184V, often emerges first. This mutation, now recognized as a keymutation in the discriminant mechanism of drug escape resistance,confers a moderate decrease in ABC susceptibility (Tisdale et al, 1997).A CNA3005 study in which a total of 562 patients randomly received AZTand 3TC with either ABC or ddI, showed a slow but steady increase in theproportion of patients carrying a TAM in the AZT and 3TC plus ABC arm.By week 48, up to 56% of the patients had at least one primerrescue-related TAM (1×TAM) over and above the rapidly induced M184Vmutation (Melby et al, 2001), illustrating the importance of preventingthe emergence of primer rescue-related resistance. Similarly, in vitropassage of AZT-resistant virus bearing the genotypic pattern of 67, 70,215 and 219 under 3TC selective pressure resulted in the selection ofthe M184V mutation and conferred cross-resistance to ABC (Tisdale et al,1997). This again highlights the concept that treating the pre-existingof primer rescue-related TAM and preventing the accumulation of primerrescue-related mutants is a pivotal step in avoiding development ofmultiple nucleoside resistance.

It has become increasingly clear that the K65R mutation quickly appearsin a very high proportion of patients who are receiving TNF or ABC.Valer et al (2004) reported that K65R increased in prevalence in theirMadrid hospital from <1% between 1997-2000 to 7% in 2003 and 12% in thefirst 4 months of 2004. The effect of the K65R mutant is exacerbated inthe presence of other mutations associated with decreased susceptibilityto ABC, 3TC, ddI and ddC (Parikh et al, 2003). Yet the simultaneousappearance of K65R of primer rescue-related TAM genotypes, althoughrarely occurring, leads to a more profound effect on the primer rescue(excision) of TNF than of AZT (Naeger et al, 2001). TNF was reported tobe active against HIV-1 with up to 3×TAMs unless the TAM clusterincluded an M41L or L210W mutation. Currently it is unclear why TAMscould reverse some of the effects of K65R, which is otherwise thought toimpede primer excision mutants with respect to susceptibility to TNF andABC. Finally, the T69D mutation was initially identified for its role incausing ddC resistance. It has also been reported to be associated witha decreased response to ddI when it occurs in combination with the T215Ymutation and other of primer rescue-related TAM genotypes.

For many years the WHO and DHHS (US Department of Health and HumanHealth Service) have recommended first-line antiretroviral therapy ontreatment naïve patients consisting of administering d4T or AZT incombination with 3TC plus nevirapine or efavirenz (Guidelines for theUse of Antiviral Retroviral Agents in HIV-1-Infected Adults andAdolescents, Jul. 14, 2003 and Mar. 23, 2004). A substantial number ofHIV-infected patients have, however, experienced treatment failure whileon their initial highly active antiretroviral therapy (HAART) regimens,suggesting that these patients are already infected with drug escapeviruses. Primer rescue-related TAM resistance mutants continue to play apivotal role in the development of drug resistance. Thus the developmentof drugs or therapeutic methods that counteract the effect of primerrescue-related TAM resistance mutants could potentiate or prolong theuse of existing NRTIs for treating treatment-naïve patients and couldalso be used to treat the primer rescue-related resistancemutant-carrying HIV infected population in a salvage therapy.

Drug Strategies for Preventing/Inhibiting Primer-Rescue Mutants

Primer rescue and discriminative mutations often appear together in thesame mutant genotype, largely due to current therapeutic strategy. AM184V mutation is representative of the family of discriminativemutants. If, however, it occurs in conjunction with primerrescue-related mutants such as M41L, D67N, K70R, L210W, T215Y/F, andK219Q/E, it plays a role in the dual resistance to AZT and 3TC (Milleret al., 1998).

These primer rescue and discriminative resistance phenotypes seem tocorrelate with different clusters of mutations in RT. For example,AZT-associated mutations comprising various permutations within M41L,E44D/A, D67N, K70R, V118I, M184V, H208Y, L210W, R211K, L214F, T215Y/F,K219Q/E and G333E, an MDR T69S mutation with 6-bp insertions and a ▴67typically exhibit primer rescue mutant activities. On the other hand,mutations at positions 65, 74, 89, 151, and 184 lead to the ability todiscriminate between NRTIs and the respective dNTP counterparts or theymay be involved in the repositioning of the primer-template complex.

In the recent article “Designing anti-AIDS drugs targeting the majormechanism of HIV-1 RT resistance to nucleoside analog drugs” (IJBCB 36(2004) 1706-1715, which is hereby incorporated by reference in itsentirety), Sarafianos et al conclude that the primer rescue (excision)mechanism could only occur before RT translocation at the N-site andfurther conclude that it has become the dominant mechanism of NRTIresistance. In the chapter entitled “Strategies for Inhibition of theExcision Reaction” (see page 1711), they propose three approaches todefeat such a resistance mechanism:

-   -   1. use of new antivirals that interfere with the productive        binding of ATP (at the P site), presumably by binding at or near        the ATP-binding site, thereby blocking the excision reaction        without affecting the forward reaction of DNA synthesis.    -   2. use of compounds that can block DNA synthesis but are somehow        resistant to excision, such as borano- or thio-substituted alpha        phosphate variants of the current NRTIs. Similarly, variants of        the current NRTIs can be engineered to reposition the        extended/terminated template/primer in a non-excisable mode, as        suggested by the poor excision capacity of the M184I/V mutants        induced by 3TC.    -   3. use of dinucleotide tetraphosphate based inhibitors to        provide bi-dentate binding at both N- and P-sites.

Each of these three proposed approaches to preventing primer rescuemechanisms of NRTI resistance is open to criticism for varioustheoretical shortcomings. For example, in the first approach ATP bindingis not required for normal RT functions. Thus, countermeasures based oninhibiting ATP or pyrophosphate binding by competition or blockage willnot prevent resistance development because the fitness of the underlyingvirus will not be compromised by such agents. In other words, resistancemutations will arise at no evolutionary cost. The abundant amount of ATPpresent in normal lymphocytes also challenges the rationale behind thisapproach.

In the second proposed approach, it seems likely that borano- orthio-substituted alpha phosphate analogues would select for thediscriminative resistant mutants, as has been seen with 3TC and FTC, andproduce HIV resistance mutants.

The third proposed approach is limited by the need for pharmacokineticuptake into the target cell of the large and highly chargedtetraphosphate dinucleotide species. This will be a severepharmaceutical and drug delivery challenge.

It is noteworthy that each of Serafaniano's approaches, includingapproach 1 which is not antiviral in itself, but presupposesco-administration of a conventional NRTI, is based on variants of thecurrent generation of NRTIs. That is, compounds that lack a 3-hydroxylfunction and therefore act as obligate chain terminators.

In contrast to the “classic” NRTIs discussed above (i.e. those lacking a3′-hydroxy function), Ohrui et al (J Med Chem (2000) 43, 4516-4525,which is hereby incorporated by reference in its entirety) describe4′-C-ethynyl HIV inhibitors:

These compounds retain the 3′-hydroxy function but nevertheless exhibitactivity against HIV-1, including a typical discriminative MDR strainbearing the A62V, V75L, F77L, F116Y and Q51 M mutations. The mechanismof action was postulated to be through affinity to the nucleosidephosphorylating kinase. It was, however, also observed that thesecompounds may be functioning as DNA chain terminators due to theirneopentyl alcohol character and the severe steric hindrance of thevicinal cis 4′ substituent, which resulted in a sharply diminishedreactivity of the 3′-hydroxy.

Kodama et al (Antimicrob Agents Chemother (2001) 1539-1546, which ishereby incorporated by reference in its entirety) describe a verysimilar set of compounds bearing a 4′-C-ethynyl group adjacent to theretained 3′-hydroxy function that were assayed in cell culture withadditional HIV resistant strains. Since Kodama et al did not prepare thetriphosphates of their compounds, they were unable to elucidate themechanism of action but infer from various circumstantial observationsthat the compounds are indeed acting as NRTIs. Kodama et al laterreported (abstract 388-T, 2003 9^(th) Conference on Retroviruses andOpportunistic Infections, which is hereby incorporated by reference inits entirety) that under the selective pressure of their 4-C-ethynylnucleoside in vitro, breakthrough resistant HIV bearing T1651 and M184Vmutations located in the RT catalytic site were found. This mutantphenotype is manifestly a discriminative type of mutation and is heavilycross resistant to 3TC. Steric conflict blocking 4-C-ethynyl nucleosideincorporation was thus implicated. This has been established with the3TC inhibitory mechanism and therefore almost certainly represents thediscriminative resistant mechanism. It therefore seems unlikely that theKodama compounds will provide guidance in addressing the mutantsfacilitating primer rescue (ATP or pyrophosphate mediated excision).

Chen et al (Biochemistry (1993) 32:6000-6002, which is herebyincorporated by reference in its entirety) conducted extensivemechanistic investigations on a structurally related series of compoundsbearing an azido group at 4′:

Chen demonstrated that RT efficiently incorporates two consecutive4′-azidothymidine monophosphate nucleotides, which terminates chainelongation. In addition, RT was also able to incorporate a first4′-azidothymidine monophosphate, followed by a native dNTP and a then asecond 4′-azidothymidine nucleotide, which also led to chaintermination. Note that both of these mechanisms resulted in a4′-azidothymidine monophosphate residing at the terminated DNA primerterminus, which is an inhibitory mechanism very reminiscent of thecurrent NRTIs. It was also apparent that the cellular (ie non-viral)polymerases α and β were each able to incorporate a single 4′-azidonucleotide, but not a second, into the nascent chain of the host DNA.These cellular polymerases then allowed the host DNA chain to elongatewith further native dNTPs and so permanently incorporated the NRTInucleotide into host DNA genes. These compounds have not been pursued inhumans because misincorporation of non-native nucleotides by cellularenzymes has clear implications in carcinogenesis. Similarly, thepharmaceutical development of the Kodama corresponding 4′-C-ethynylcompounds was stopped, allegedly due to severe toxicity in higherorganisms.

EP 341 911 describes an extensive family of 3′-C-hydroxymethylnucleosides of the formula

and proposes their use predominantly against herpesviruses such as CMV,but also against retroviruses. WO92/06201 also discloses a similar setof compounds and indications.

U.S. Pat. No. 5,612,319 (which is hereby incorporated by reference inits entirety) discloses the retroviral activity of 2′-3′dideoxy-3′-C-hydroxymethylcytosine against wild type HIV-1_(IIIB) andthe simian equivalent, SIV-1, in an acute cynomolgus monkey model of HIVinfection. This publication proposes the use of the compound as apost-exposure prophylaxis agent, especially against needle-stickinjuries. Post exposure prophylaxis implies that the active ingredientis immediately administered to people such as medical personnel, whohave unwittingly jabbed themselves with a potentially HIV-infectedsyringe. In order to ensure rapid treatment of an understandably shockedhealth care professional, a self administered spring-loaded syringe,such as are used for antidotes to chemical and biological warfare, is apreferred administration route.

The intention of post-exposure prophylaxis is to prevent the infectionfrom establishing itself rather than treating an on-going infection. Assuch, it was intended that treatment was to be carried out for a shorttime period such as 24-48 hours, using extremely high doses of thecompound. This publication states that because of the discrete timeperiod of administration, transient toxicity is acceptable because oneis trying to prevent an incurable disease. The post-exposureprophylactic method described in U.S. Pat. No. 5,612,319 has never beentried in humans—indeed to our knowledge 2′-3′dideoxy-3′-C-hydroxymethylcytosine has not been administered to humansat all.

In 1994 when the application granting as U.S. Pat. No. 5,612,319 wasfiled, multi-resistant HIV as it is known today had not arisen in anycogent form. Today's multi-resistant HIV has primer rescue mutationsinduced by and accumulated from many years of selective pressure fromNRTI therapy. In other words, the HIV and especially the RT existent atthe time these patents were granted was structurally and mechanisticallyvery different from today's viruses.

International patent application PCT/EP2005/057196, which wasunpublished at the priority date of the present application, disclosesthe use of 2′,3′-dideoxy-3′-hydroxymethylcytosine and prodrugs thereofin the treatment of HIV escape mutants.

It is believed that 2′,3′-dideoxy-3′-C-hydroxymethylcytosine isphosphorylated to the corresponding 5′-triphosphate by cellular enzymes.The heavily mutated RT of multiresistant HIV, in particular primerrescue-related mutant RT, incorporates this triphosphate as the5′-(2′,3′-dideoxy-3′-C-hydroxymethylcytosine) monophosphate into thenascent DNA chain.

Conventional NRTIs act as obligate chain terminators, terminating DNAsynthesis at the N-site, and are thus susceptible to the above describedATP- or pyrophosphate mediated primer rescue (excision) mechanism uniqueto mutiresistant HIV. In contrast,5′-(2′,3′-dideoxy-3′-C-hydroxymethylcytosine) monophosphate does not actas an obligate chain terminator, but rather allows an additional residueto be covalently attached to the 3′ hydroxymethyl function of the5′-(2′,3′-dideoxy-3′-C-hydroxymethylcytosine) monophosphate. This thenpromotes the RT to undergo the necessary transformational change totranslocate itself into the P-site for the next round of polymerization.Preliminary evidence suggests that this attached terminal residue is anative nucleotide rather than a further5′-(2′,3′-dideoxy-3′-C-hydroxymethyl cytosine) monophosphate.

Importantly, data suggests that the last incorporated,non-2′3′-dideoxy-3′-C-hydroxymethylcytosine nucleotide is not amenableto the further addition of nucleotides by the mutated reversetranscriptase. That is, chain termination appears to occur one basebeyond the NRTI of the invention rather than at the NRTI. Furthermore,following the incorporation of 2′,3′-dideoxy-3′-hydroxymethylcytosine,the RT appears to successfully translocate to the P-site in order toaccept the next incoming nucleotide. This evidence suggests that2′,3′-dideoxy-3′-hydroxymethylcytosine, in conjunction with a primerrescue-related mutated RT, achieves a form of chain termination which isnot amenable to ATP- or pyrophosphate induced excision. As aconsequence, 2′,3′-dideoxy-3′-hydroxymethylcytosine allows effectivetreatment of HIV infections that are non-responsive to current drugregimes.

The inhibitory mechanism discussed immediately above is thusfundamentally different from the chain termination mechanism of the4′-substituted nucleosides of Chen et al (see above), which allowsseveral nucleotides to be incorporated after the incorporated4-substituted compound. Firstly, the Chen mechanism dramaticallyenhances the risk of “readthrough.” That is, the DNA polymerasecontinues to follow the coding strand and continues to add the codedresidues to the normal stop codon, thereby misincorporating the abnormalnucleoside within the DNA strand. Antiviral efficacy can be lost,however, when a viral DNA strand is constructed by the viral polymerase(i.e. RT) since the readthrough construct may still be viable,notwithstanding the misincorporated 4′-substituted nucleoside. Moreimportantly, if the 4′-substitued nucleoside is readthrough by acellular (i.e. host) polymerase, as Chen describes, the resultingconstruct thereafter represents a teratogen and dramatically increasesthe risk of cellular damage and cancer.

The Chen compounds additionally require the addition of a second4′-substituted nucleotide, either immediately adjacent to the firstmis-incorporated 4′-substituted nucleotide (i.e. X-X) or interspersed byone native nucleotide (i.e. X—N—X). In practice this means that thenucleotide at the last position of the primer terminus is the non-native(i.e. drug) nucleotide. This is an analogous situation to the case ofclassic NRTIs (i.e. those lacking a 3-hydroxy group) chain termination.Here, the NRTI nucleotide also resides at the last position of theprimer terminus where, as discussed above, it is susceptible to ATP orpyrophosphate mediated excision.

Multiple units of the Chen 4′-substituted nucleotide are needed in orderfor it to work as an efficient RT inhibitor. As a consequence, thedrug's effectiveness depends on the sequence of the reading strand. Forexample, if the Chen compound is a thymidine analogue it will have thebest affinity if the reading strand has an AA or A-N-A sequence. Here,the drug would be efficient and effective in terminating DNA synthesis.But if the reading strand's sequence does not contain abundant recitalsof the AA or A-N-A sequence, the Chen drug will be less able toterminate DNA synthesis, at a given concentration. Since an AA doubletor an A-N-A triplet is far less common in the genome than a singlet A,the Chen drug will be far less efficient than other NRTIs that do nothave a multiple unit requirement.

Mauldin et al Bioorganic and Medicinal Chemistry 1998 6:577-585discloses a number of 2′,3′-dideoxy-3′-hydroxymethylcytosine prodrugs.Of particular note is the fact that the authors found that prodrugsinvolving substituents at the alcohol positions resulted in a decreasein antiviral activity in virtually all of their assays. It is an objectof the present invention to provide novel prodrugs of2′,3′-dideoxy-3′-hydroxymethylcytosine, of use in the treatment of HIV,and in particular in the treatment of HIV escape mutants.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with a first aspect of the invention, there are providednovel compounds of the formula I:

wherein:R¹ is independently H, —OR³, —NHR⁴; C₁-C₄ alkyl;

-   -   or, when n is 2, adjacent R¹ together define an olefinic bond;

R² is H;

-   -   or when the gem R¹ is C₁-C₄ alkyl, that R² may also be C₁-C₄        alkyl;    -   or when the gem R¹ is —OR³, that R² may also be —C(═O)OH or a        pharmaceutically acceptable ester thereof;        R³ is independently H, or a pharmaceutically acceptable ester        thereof;        R⁴ is independently H or a pharmaceutically acceptable amide        thereof;        R⁵ is H, —C(═O)R⁷, or an amide-bound L-amino acid residue;

R⁶ is H;

-   -   or R⁵ and R⁶ together define the imine ═CR⁸R^(8′);        R⁷ is C₁-C₆ alkyl, C₀-C₃alkylcycyl;        R⁸ and R^(8′) are independently H, C₁-C₆ alkyl, C₀-C₃alkylcycyl;    -   or R⁸ is H and R^(8′) is —NR⁹R^(9′);        R⁹ and R^(9′) are independently H, C₁-C₆ alkyl, C₀-C₃alkylcycyl;    -   or R⁹ and R^(9′) together with the N atom to which they are        attached define a saturated 5 or 6 membered ring;        n is 1, 2 or 3;        and pharmaceutically acceptable salts thereof.

According to one embodiment of the invention, n is 1 and the compoundshave the general formula:

Favoured variants for R1:R2 in this embodiment include H:H, H:OH or apharmaceutically acceptable ester thereof, and Me:Me. Particularlyfavoured variants of this embodiment have H as R⁵ and R⁶.

An alternative embodiment of the invention has n=2, thereby producingcompounds of the general formula:

In this embodiment favoured variants for R¹a:R¹b:R²a:R²b include

H:H:H:H

H:H.H:OH or a pharmaceutically acceptable ester thereofH:H:H:NH₂ or a pharmaceutically acceptable amide thereofH:OH or a pharmaceutically acceptable ester thereof: H:HH:NH₂ or a pharmaceutically acceptable amide thereof: H:H

Me:Me:H:H H:H:Me:Me H:OH:H:OH H:C═C:H

Particularly favoured variants of this embodiment have H as R⁵ and R⁶.

A further embodiment of the invention has n equal to 3, with the generalformula:

Favoured variants of R¹a:R²a:R²a:R²b:R¹c:R²c include

H:H:H:H:H:H H:H:Me:Me:H:H H:H:OH:H:H:H H:H:OH:COOH:H:H H:H:H:H:H:NH₂

or a pharmaceutically acceptable ester or amide thereof.

Particularly favoured variants of this embodiment have H as R⁵ and R⁶.

Certain embodiments of the invention have a modified base, ie R⁵ and/orR⁶ are other than hydrogen. One such embodiment are imines, wherein R⁵and R⁶ together define the imine ═CR⁸R^(8′). Typically R⁸ and R^(8′)will each be the same alkyl group, but asymmetric R⁸/R^(8′) variants arealso within the scope of the invention. Representative imines withinthis embodiment include:

-   -   ═CHN(CH₃)₂    -   ═CHN(ipr)₂    -   ═CHN(pr)₂

Alternatively R⁸ and R^(8′) can together define a cyclic group such aspyrrolidine, piperidine, piperazine, N-methyl piperazine or morpholine,Representative imines thus include:

-   -   ═CHN(CH₂)₄    -   ═CHN(CH₂)₅    -   ═CHN(CH₂)₆    -   ═CHN(CH₂CH₂)₂O

It is currently preferred that R⁵ and R⁶ are H.

Other embodiments of the invention wherein R⁵ and R⁶ are other than Hinclude amides, such as L-amino acid amides, such as Ile, Val, Leu orPhe amides. Alternative amides include alkyl amides such as C₁-C₆ alkylamides, for example those wherein R⁵ is C(═O)CH₃, C(═O)CH₂CH₃ orC(═O)C(CH₃)₃. Other amides include C(═O)CO—C₃alkylaryl amides, such asC(═O)Ph or C(═O)Bz.

Currently preferred embodiments include the compounds of formula Idenoted

-   2-(4-amino-2-oxo-2H-pyrimidin-1-yl)-octahydro-1,5,10-trioxacyclopenta-cyclodecene-6,9-dione;    or-   2-(4-amino-2-oxo-2H-pyrimidin-1-yl)-octahydro-1,5,11-trioxa-cyclopentacycloundecene-6,10-dione;    or a pharmaceutically acceptable salt thereof. These compound    release innocuous by-products upon hydrolysis in vivo, such as    succinic or glutaric acid.

Although not wishing to be bound by theory it is believed that thecompounds of the invention, or active metabolites thereof, are activeagainst the reverse transcriptase of retroviruses such as HIV-1, HIV-2,HTLV and SIV.

Accordingly a further aspect of the invention provides methods for theprophylaxis or treatment of retrovirus infections in humans or animalscomprising the administration of a compound of the formula I, or apharmaceutically acceptable salt thereof. Typically the administrationis oral.

A further aspect of the invention provides the use of compounds of theformula I, or a pharmaceutically acceptable salt thereof in themanufacture of a medicament for the treatment or prophylaxis ofretroviral infections in humans or animals. Typically the medicament isin a form adapted for oral administration.

Another embodiment of the invention provides a method for inhibiting theemergence or propagation of HIV primer rescue mutants that are able toremove a chain-terminating NRTI nucleotide incorporated into an HIVprimer/template complex where the removal is effected by anATP-dependent or pyrophosphate dependent excision mechanism. The methodcomprises the simultaneous or sequential administration to an individualinfected with HIV an effective amount of the compound of the inventionand at least one chain terminator NRTI which induces primer rescuemutants.

Conventional NRTIs act as obligate chain terminators, terminating DNAsynthesis at the N-site, and are thus susceptible to the above describedATP- or pyrophosphate mediated primer rescue (excision) mechanism uniqueto mutiresistant HIV. In contrast, preliminary evidence suggests thatthe compounds of the invention do not act as an obligate chainterminator, but rather allows an additional residue to be covalentlyattached to the 3′ hydroxymethyl function of the5′-(2′,3′-dideoxy-3′-C-hydroxymethylcytosine) monophosphate. This thenpromotes the RT to undergo the necessary transformational change totranslocate itself into the P-site for the next round of polymerization.Preliminary evidence based on the sequence of the template presentedbelow suggests that this attached terminal residue is a nativenucleotide.

The multiresistant HIV typically able to be treated or preventedaccording to the invention will typically have an RT bearing a geneticpattern comprising at least one of

-   -   (a) M41, ±D67, L210 and T215;    -   (b) D67, K70 and K219;    -   (c) T69S-XX or    -   (d) ▴67        where XX represents an addition to the RT sequence of any two        natural amino acids and ▴67 represent the amino acid deletion at        codon 67.

Although the above 4 genetic patterns are believed to represent theessential basis of the excision drug escape phenotype, t will beapparent that the mutants treated or prevented by the use of theinvention will typically comprise additional mutations in the RT geneand elsewhere, often at least three mutations in the RT gene.

Generally, but not exclusively, the cluster M41, D67, L210 and T215 willoften comprise M41L, D67N, L210W and T215Y or T215F. Optionally, theclusters immediately above may further comprises at least one furthermutation at position E44, K70, V118, H208, R211K, L214, K219 or G333.

The clusters immediately above may further comprise at least oneadditional mutation at position ▴67, T69, E203, L210, D218, H221, D223or L228.

Generally, but not exclusively, the cluster D67, K70 and K219 comprisesD67N, K70R and K219Q or K219E.

Optionally, the cluster D67, K70 and K219 may further comprise at leastone additional mutation at position M41, E44, V118, H208, L210, R211K,L214, T215, or G333.

In addition, the cluster D67, K70 and K219 optionally further comprisesat least one additional mutation at position ▴67, T69, E203, L210, D218,H221, D223 or L228.

Generally, but not exclusively, the cluster T69S-XX may further compriseat least one additional mutation at position M41, E44, D67, K70, V118,H208, L210, R211K, L214, T215, K219 or G333.

Optionally, the cluster T69S-XX may further comprise at least oneadditional mutation at position ▴67, T69, E203, L210, D218, H221, D223or L228.

Generally, but not exclusively, the cluster ▴67 may further comprise atleast one additional mutation at position M41, E44, D67, K70, V118,H208, L210, R211K, L214, T215, K219 or G333.

Optionally, the cluster ▴67 may further comprise at least one additionalmutation at position T69, T69S+XX, E203, L210, D218, H221, D223 or L228.Optionally, the reverse transcriptase may further bear at least onediscriminative mutation at position K65, L74, M184 or Q151, especiallyK65R, L74V or M184V or Q151M.

Typically, the cluster of discriminative mutants may be linked with atleast one additional mutation at position A62, V75, F77, Y115 or F116.

Among the HIV strains able to be treated by the invention aremultiresistant HIV strains whose RT has mutations that encourage ATP- orpyrophosphate-mediated primer rescue (excision) of chain terminatingNRTI nucleotides and which has arisen within the patient as a result ofprevious HlV-treatment with at least one antiviral selected fromzudovudine (AZT, ZDV), stavudine (d4T), zalcitabine (ddC), didanosine(ddI), abacavir, (ABC), lamivudine (3TC), emtricitabine (FTC), adefovir(ADV), entacavir (BMS 200475) alovudine (FLT), tenofovir disoproxilfumarate (TNF), amdoxavir (DAPD), D-d4FC (DPC-817), -dOTC(SPD754),SPD-756, racivir, D-FDOC or GS7340.

Alternatively, the HIV strains are those found in patients who havereceived such a resistant or multiresistant HIV strain directly orindirectly from another individual who had themselved induced aresistant or multiresistant HIV strain by sustained treatment with atleast one antiviral from the above list of NRTI antivirals. Frequentlythe mulitresistant HIV strains contain at least three mutations in theviral RT as compared to wildtype.

It will thus be apparent that the methods and composition of theinvention may be used as an add-on to current antiretroviral therapies,such as HAART, or in some cases as a rescue or salvage therapy. Thiswill typically be the case where the multiresistant HIV has been inducedin the actual patient by that patient's earlier antiretroviral drugtreatment history. Alternatively, the methods and compositions of theinvention will constitute a first line therapy, typically in patientswhose primary HIV infection occurred with an already-mutatedmultiresistant strain. The following antiviral drugs often induce suchmultiresistant HIV strains having RT primer rescue mutations whichencourage ATP- or pyrophosphate-mediated excision of chain terminatingNRTI nucleotides:

zudovudine, lamivudine or the combined dosage forms Combivir orTrizivir;lamivudine, abacavir or the combined dosage form Epzicom;tenofovir, emtricitabine or the combined dosage form Truvada.

While these drugs frequently induce such multiresistant HIV strains,this drug list is not exclusive.

It is therefore apparent that the compound of the invention isadministered in order to prevent the emergence of one or moremultiresistant HIV strains having RT primer rescue mutations thatencourage ATP- or pyrophosphate-mediated excision of chain terminatingNRTI nucleotides. This prevention occurs even when NTRI drugs whichinduce such mutations are administered concomitantly.

A third aspect of the invention provides a pharmaceutical composition inunit dosage form comprising the compound of formula I and at least onechain terminator NRTI, where upon sustained dosing with the NRTIinduces, HIV RT primer rescue mutations which encourage ATP-dependent orpyrophosphate-dependent excision of incorporated NRTI monophosphate fromthe 3′-terminus of the primer/template complex and allows resumption ofDNA synthesis.

Preferred embodiments of the pharmaceutical composition of the inventionand the method of the invention include those where the NRTI is selectedfrom zudovudine (AZT, ZDV), stavudine (d4T), zalcitabine (ddC),didanosine (ddI), abacavir, (ABC), lamivudine (3TC), emtricitabine(FTC), adefovir (ADV), entacavir (BMS 200475), alovudine (FLT),tenofovir disoproxil fumarate (TNF), amdoxavir (DAPD), D-d4FC (DPC-817),-dOTC(SPD754), SPD-756, racivir, D-FDOC or GS7340 and combinationsthereof.

Particularly preferred embodiments include those where the NRTI isselected from: zidovudine, stavudine, didanosine, lamivudine, abacavir,tenofovir, emtricitabine or combinations thereof.

Experience with HIV drugs, and HIV reverse transcriptase inhibitors inparticular, has further emphasized that sub-optimal pharmacokinetics andcomplex dosage regimes quickly result in inadvertent compliancefailures. This in turn means that the 24 hour trough concentration(minimum plasma concentration) for the respective drugs in an HIV regimefrequently falls below the IC₉₀ or ED₉₀ threshold for large parts of theday. It is considered that a 24 hour trough level of at least the IC₅₀,and more realistically, the IC₉₀ or ED₉₀ is essential to slow down thedevelopment of drug escape mutants.

The compounds of the invention are typically administered to the patientat a dose commensurate with the expectation of a sustained andprotracted antiretroviral treatment. The treatment regimen thus aims toensure a defined drug level, yet to avoid toxicity, although the use ofcompounds of formula I in a high-dose, acute, post-exposure prophylaxistreatment can tolerate some transient toxicity is acceptable. Thecompounds of formula I are typically administered in ranges of 1-25mg/kg/day, preferably less than 10 mg/kg/day, preferably in the range of0.05-0.5 mg/kg/day. The appropriate dosage will depend upon theindications and the patient, and is readily determined by conventionalanimal drug metabolism and pharmacokinetics (DMPK) or clinical trialsand in silico prediction software.

The unit dosage pharmaceutical compositions of the invention havecorresponding amounts of the compound of formula I, typically scaled fora 60 kg or 75 kg adult, and are optionally divided once, twice or threetimes for a QD, BID or TID dosage regime. If the therapeutic dose is inthe range of 0.05-0.5 mg/kg/day, then a clinical QD dose per person perday would be 3 mg-30 mg for a 60 kg adult or 3.75-37.5 mg for a 75 kgadult. Dosage and regiment restrictions of the additional conventionalNRTI in the combined dosage unit pharmaceutical composition aspect ofthe invention may necessitate QD, BID or TID dosing.

The current invention includes pharmaceutically acceptable salts such assalts of organic acids, especially carboxylic acids, including but notlimited to acetate, trifluoroacetate, lactate, gluconate, citrate,tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate,aspartate, benzoate, butyrate, digluconate, cyclopentanate,glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate,fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate,pivalate, proprionate, tartrate, lactobionate, pivolate, camphorate,undecanoate and succinate. Also included are the salts of organicsulphonic acids such as methanesulphonate, ethanesulphonate,2-hydroxyethane sulphonate, camphorsulphonate, 2-napthalenesulphonate,benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate.The acceptable salts also include those from inorganic acids such ashydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate,hemisulphate, thiocyanate, persulphate, phosphoric and sulphonic acids.

The current invention extends to active agents that are hydrates,solvates, complexes and other physical forms releasing the compound offormula I.

While it is possible for the active agent to be administered alone, itis preferable to present it as part of a pharmaceutical formulation.Such a formulation will comprise the compound of formula I active agenttogether with one or more acceptable carriers/excipients and optionallyother therapeutic ingredients. The carrier(s) must be acceptable in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient.

The formulations include those suitable for rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal)administration. Preferably the formulation is an orally administeredformulation. The formulations may conveniently be presented in unitdosage form, e.g. tablets and sustained release capsules, and may beprepared by any methods well known in the art of pharmacy.

Such well known methods include the step of bringing the compound offormula I active agent into association with the carrier. In general,the formulations are prepared by uniformly and intimately bringing theactive agent into association with liquid carriers or finely dividedsolid carriers or both, and then shaping the product, if necessary. Theinvention extends to methods for preparing a pharmaceutical compositioncomprising bringing a compound of formula I or its pharmaceuticallyacceptable salt in conjunction or association with a pharmaceuticallyacceptable carrier or vehicle. If the manufacture of pharmaceuticalformulations involves intimate mixing of pharmaceutical excipients andthe active ingredient is in a salt form, then it is often preferred touse excipients which are non-basic in nature, i.e. either acidic orneutral.

The formulations for oral administration of the present invention may bepresented as discrete units such as capsules, cachets or tablets, eachcontaining a predetermined amount of the active agent. Alternativelythey can be presented as a powder or granules; as a solution or asuspension of the active agent in an aqueous liquid or a non-aqueousliquid, or as an oil-in-water liquid emulsion or a water-in-oil liquidemulsion, as a bolus, etc.

With regard to compositions for oral administration (e.g. tablets andcapsules), the term “suitable carrier” includes vehicles such as commonexcipients, for example binding agents such as syrup, acacia, gelatin,sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose,ethylcellulose, sodium carboxymethylcellu lose,hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers,for example corn starch, gelatin, lactose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride andalginic acid; and lubricants such as magnesium stearate, sodium stearateand other metallic stearates, glycerol stearate stearic acid, siliconefluid, talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring or the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredient. Compressed tablets may be prepared bycompressing in a suitable machine the active agent in a free flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent. Moulded tablets may be made by moulding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may optionally be coated or scored and may be formulated soas to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured base, usually sucrose andacacia or tragacanth; pastilles comprising the active agent in an inertbase such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active agent in a suitable liquid carrier.

‘C₁-C₆alkyl’ (also abbreviated as C₁-C₆alk, or used in compoundexpressions such as C₁-C₆alkyloxy etc) as applied herein is meant toinclude straight and branched chain aliphatic carbon chains such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl,isopentyl, hexyl, heptyl and any simple isomers thereof. The alkyl groupmay have an unsaturated bond. Additionally, any C atom in C₁-C₆alkyl mayoptionally be substituted by one, two or where valency permits threehalogens and/or substituted or the alkylene chain interrupted by aheteroatom S, O, NH. If the heteroatom is located at a chain terminusthen it is appropriately substituted with one or 2 hydrogen atoms.C₁-C_(n)alkyl has the corresponding meaning to C₁-C₆alkyl adjusted asnecessary for the carbon number.

‘CO—C₃alkylaryl’ as applied herein is meant to include an aryl moietysuch as a phenyl, naphthyl or phenyl fused to a C₃-C₇cycloalkyl forexample indanyl, which aryl is directly bonded (i.e. C₀) or through anintermediate methyl, ethyl, propyl, or isopropyl group as defined forC₁-C₃alkylene above. Unless otherwise indicated the aryl and/or itsfused cycloalkyl moiety is optionally substituted with 1-3 substituentsselected from halo, hydroxy, nitro, cyano, carboxy, C₁-C₆alkyl,C₁-C₆alkoxy, C₁-C₆alkoxyC₁-C₆alkyl, C₁-C₆alkanoyl, amino, azido, oxo,mercapto, nitro C₀-C₃alkylcarbocyclyl, C₀-C₃alkylheterocyclyl. “Aryl”has the corresponding meaning, i.e. where the C₀-C₃alkyl linkage isabsent.

‘C₀-C₃alkylC₃C₇cycloalkyl’ as applied herein is meant to include aC₃-C₇cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl or cycloheptyl, which cycloalkyl is directly bonded (i.e.C₀alkyl) or through an intermediate methyl, ethyl or proyl group asdefined for C₁-C₃alkylene above. The cycloalkyl group may contain anunsaturated bond. Unless otherwise indicated the cycloalkyl moiety isoptionally substituted with 1-3 substituents selected from halo,hydroxy, nitro, cyano, carboxy, C₁-C₆alkyl, C₁-C₆alkoxy,C₁-C₆alkoxyC₁-C₆alkyl, C₁-C₆alkanoyl, amino, azido, oxo, mercapto, nitroC₀-C₃alkylcarbocyclyl, C₀-C₃alkylheterocyclyl.

‘C₀-C₃alkylcarbocyclyl’ as applied herein is meant to includeC₀-C₃alkylaryl and CO—C₃alkylC₃-C₇cycloalkyl. Unless otherwise indicatedthe aryl or cycloalkyl group is optionally substituted with 1-3substituents selected from halo, hydroxy, nitro, cyano, carboxy,C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkoxyC₁-C₆alkyl, C₁-C₆alkanoyl, amino,azido, oxo, mercapto, nitro, CO—C₃alkylcarbocyclyl and/orC₀-C₃alkylheterocyclyl. “Carbocyclyl” has the corresponding meaning,i.e. where the C₀-C₃alkyl linkage is absent ‘C₀-C₃alkylheterocycylyl’ asapplied herein is meant to include a monocyclic, saturated orunsaturated, heteroatom-containing ring such as piperidinyl,morpholinyl, piperazinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl,thiazinolyl, isothiazinolyl, thiazolyl, oxadiazolyl, 1,2,3-triazolyl,1,2,4-triazolyl, tetrazolyl, furanyl, thienyl, pyridyl, pyrimidyl,pyridazinyl, pyrazolyl, or any of such groups fused to a phenyl ring,such as quinolinyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl,benzothiazinolyl, benzisothiazinolyl, benzothiazolyl, benzoxadiazolyl,benzo-1,2,3-triazolyl, benzo-1,2,4-triazolyl, benzotetrazolyl,benzofuranyl, benzothienyl, benzopyridyl, benzopyrimidyl,benzopyridazinyl, benzopyrazolyl etc, which ring is bonded directly i.e.(C₀), or through an intermediate methyl, ethyl, propyl, or isopropylgroup as defined for C₁-C₃alkylene above. Any such non-saturated ringshaving an aromatic character may be referred to as heteroaryl herein.Unless otherwise indicated the hetero ring and/or its fused phenylmoeity is optionally substituted with 1-3 substituents selected fromhalo, hydroxy, nitro, cyano, carboxy, C₁-C₆alkyl, C₁-C₆alkoxy,C₁-C₆alkoxyC₁-C₆alkyl, C₁-C₆alkanoyl, amino, azido, oxo, mercapto,nitro, C₀-C₃alkylcarbocyclyl, C₀-C₃alkylheterocyclyl. “Heterocyclyl” and“Heteroaryl” have the corresponding meaning, i.e. where the C₀-C₃alkyllinkage is absent.

Typically heterocycyl and carbocyclyl moieties within the scope of theabove definitions are thus a monocyclic ring with 5 or especially 6 ringatoms, or a bicyclic ring structure comprising a 6 membered ring fusedto a 4, 5 or 6 membered ring.

Typical such groups include C₃-C₈cycloalkyl, phenyl, benzyl,tetrahydronaphthyl, indenyl, indanyl, heterocyclyl such as fromazepanyl, azocanyl, pyrrolidinyl, piperidinyl, morpholinyl,thiomorpholinyl, piperazinyl, indolinyl, pyranyl, tetrahydropyranyl,tetrahydrothiopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl,pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl,pyrimidinyl, pyrazinyl, pyridazinyl, tetrazolyl, pyrazolyl, indolyl,benzofuranyl, benzothienyl, benzimidazolyl, benzthiazolyl, benzoxazolyl,benzisoxazolyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl,tetrahydroisoquinolinyl, quinazolinyl, tetrahydroquinazolinyl andquinoxalinyl, any of which may be optionally substituted as definedherein.

The saturated heterocycle moiety thus includes radicals such aspyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl,morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, piperazinyl,indolinyl, azetidinyl, tetrahydropyranyl, tetrahydrothiopyranyl,tetrahydrofuranyl, hexahydropyrimidinyl, hexahydropyridazinyl,1,4,5,6-tetrahydropyrimidinylamine, dihydro-oxazolyl,1,2-thiazinanyl-1,1-dioxide, 1,2,6-thiadiazinanyl-1,1-dioxide,isothiazolidinyl-1,1-dioxide and imidazolidinyl-2,4-dione, whereas theunsaturated heterocycle include radicals with an aromatic character suchas furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl,pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, tetrazolyl,thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl,indolizinyl, indolyl, isoindolyl. In each case the heterocycle may becondensed with a phenyl ring to form a bicyclic ring system.

The compounds of formula I include certain pharmaceutically acceptableesters or amides. Representative esters thus include carboxylic acidesters in which the non-carbonyl moiety of the carboxylic acid portionof the ester grouping is selected from straight or branched chain alkyl(for example, methyl, n-propyl, t-butyl, or n-butyl), cycloalkyl,alkoxyalkyl (for example, methoxymethyl), aralkyl (for example benzyl),aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl,optionally substituted by, for example, halogen, C₁₋₄ alkyl, or C₁₋₄alkoxy) or amino); sulphonate esters, such as alkyl- or aralkylsulphonyl(for example, methanesulphonyl); amino acid esters (for example, L-valylor L-isoleucyl); and mono-, di-, or tri-phosphate esters. In suchesters, unless otherwise specified, any alkyl moiety presentadvantageously contains from 1 to 18 carbon atoms, particularly from 1to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Anycycloalkyl moiety present in such esters advantageously contains from 3to 6 carbon atoms. Any aryl moiety present in such esters advantageouslycomprises a phenyl group, optionally substituted as shown in thedefinition of carbocycylyl above.

Pharmaceutically acceptable esters thus include C₁-C₂₂ fatty acidesters, such as acetyl, t-butyl or long chain straight or branchedunsaturated or omega-6 monounsaturated fatty acids such as palmoyl,stearoyl and the like.

Alternative aryl or heteroaryl esters include benzoyl, pyridylmethyloyland the like any of which may be substituted, as defined in carbocyclylabove. Preferred pharmaceutically acceptable esters include aliphaticL-amino acid esters such as leucyl, isoleucyl and especially valyl.Additional preferred amino acid esters include the 2-O-AA-C₃-C₂₂ fattyacid esters described in WO99 09031, where AA is an aliphatic amino acidester, especially those derived from L-lactic acid and L-valyl.

Pharmaceutically acceptable amides include those derived from C₁-C₂₂branched or straight chain aminoalkyl optionally including 1 to 3unsaturations and/or optionally substituted with the substituentsdefined in carbocycylyl above, or anilines or benzylamines. Preferredamides include those formed from reaction of the amine with a C₁-C₄straight or branched chain alkanoic acid. Other pharmaceuticallyacceptable amides of amine functions correspond to the amides of thecarboxylic acids preferred for the esters indicated above.

Synthesis

The compounds of the invention are typically synthesized from adifferentially protected bis-4,5-hydroxymethyltetrahyrdofuran derivativeprepared analogously to Svansson L. et al. in J. Org. Chem. (1991) Vol56: 2993-2997, as outlined in Scheme 1:

In Scheme 1, chiral epoxy alcohol 1 is readily prepared using Sharplessoxidation as shown in J Org Chem 1987, 52, 2596. Rs is a conventionalhydroxyl protecting group such as those discussed below, for examplepara-bromobenzyl. Epoxy alcohol 1 is regioselectively alkylated at C-3,for example with allyl magnesium bromide in dimethyl ether at −50degrees C. Chromatography, for example with silica gel, separates thedesired isomer 2, optionally after a differentiation step in which thevicinyl hydroxyls of the non-desired regioisomer are cleaved with anoxidizing agent such as sodium periodate. The primary hydroxyl group in2 is protected with a further hydroxyl protecting group, for examplebenzoylation with benzoyl chloride in pyridine at 0 degrees C. producinga differentially protected 3. Cis-hydroxylation of the olefinic bondusing a catalytic amount of osmium tetroxide and N-methylmorpholineN-oxide as reoxidant, as described in Tet. Lett 1976 17 1973 yields thecorresponding di-alcohol which in turn is cleaved with an oxidizingagent such as sodium periodate in an organic solvent such aqueoustetrahydrofuran. The thus-produced unstable furanose is deblocked with aalcohol/acid such as 0.5% w/w methanol in hydrochloric acid to give thedifferentially protected bis-4,5-hydroxymethyltetrahydrofuranintermediate 4.

It may be desirable to manipulate the protecting groups Rs and Rs1 (ieto remove and reprotect with a further hydroxyl protecting group,thereby to optimize the ease of selective removal of a selected one ofthe protecting groups and not the other in later steps. The differentialprotecting groups in 4 are thus selected so as to enable selectiveremoval of one such protecting group and acylation of the thus-exposedhydroxyl function as shown below in Schemes 2 and 3. Many such pairs ofdifferentially selectable hydroxyl protecting groups are known, forexample the O-protecting groups disclosed in Greene, “Protective GroupsIn Organic Synthesis,” (John Wiley & Sons, New York (1981)).

Hydroxy-protecting groups thus comprise ethers such as methyl ether orsubstituted methyl ethers, for example, methoxymethyl (MOM),benzyloxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl (MEM),2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl,2-(trimethylsilyl)ethoxymethyl, terohydropyranyl (THP),3-bromotetrahydropyranyl, tetrahydrothiopyranyl,4-methoxytetrahydro-pyranyl, 4-methoxytetrahydrothiopyranyl S,S dixido,tetrahydrofuranyl and tetrahydrothiofuranyl. Ethyl ethers include1-ethoxyethyl, 1-methyl-1-methoxyethyl, 1-(isopropoxy)ethyl,2,2,2-trichloroethyl and 2-(phenylselenyl)ethyl. Other ethers includet-butyl, allyl, cinnamyl, p-chlorophenyl and benzyl ethers such asunsubstituted benzyl, p-methoxybenzyl, o-nitrobenzyl, p-nitrobenzyl,p-halobenzyl and p-cyanobenzyl. Other ethers include 3-methyl-2-picolylN-oxido, diphenylmethyl, 5-dibenzosuberyl, triphenylmethyl, alphanaphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl,p(p′-bromophenacyloxy)phenyldiphenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl(tritylone) andbenzisothiazolyl S,S dioxodo. Silyl ethers include trimethylsilyl (TMS),triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl (TBDMS),(triphenylmethyl)dimethylsilyl, t-butyldiphenylsilyl,methyldiisopropylsilyl, methyldi-t-butylsilyl, tribenzylsilyl,tri-p-xylylsilyl, triisopropylsilyl and tripenylsilyl. Alternativehydroxyl protecting groups include esters, such as the formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,p-(P)-phenylacetate, 3-phenylpropionate, 3-benzoylpropionate,isobutyrate, monosuccinate, 4-oxopenatanoate (levinulate), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, (E)-2-methyl-2-butenoate(tigloate) and benzoates such as the unsubstituted, oro-(dibromomethyl)-, o-(methoxycarbonyl)-, p-phenyl-,2,4,6-trimethyl-(mesitate) or p-(P)-benzoates, or alpha-naphthoate.Carbonate hydroxyl protecting groups include the methyl, ethyl,2,2,2-trichloroethyl, isobutyl, vinyl, allyl, cinnamyl, p-nitrophenyl,benzyls such as the unsubstituted, p-methoxy-, 3,4-dimethoxy-, o-nitro-or p-nitrobenzyls, or S-benzyl thiocarbonate. Miscellaneous hydroxylprotecting groups include N-phenylcarbamate, N-imidazolylcarbamate,borate, nitrate, N,N,N,N-tetramethylphosphorodiamidate and2,4-dinitrophenylsulfenate. Greene provides extensive reactivity chartsto facilitate is selecting complementary pairs of differentialprotecting groups.

Representative hydroxyl protecting groups include those in the examples,and ethers such as t-butyl and other lower alkyl ethers, such asisopropyl, ethyl and especially methyl, benzyl and triphenylmethyl;tetrahydropyranyl ethers; substituted ethyl ethers, for example,2,2,2-trichloroethyl; silyl ethers, for example, trimethylsilyl,t-butyldimethylsilyl and t-butyldiphenylsilyl; and esters prepared byreacting the hydroxyl group with a carboxylic acid, for example,acetate, propionate, benzoate and the like.

The differentially protected bis-4,5 hydroxmethyltetrahydrofuran 4 isthen condensed with a silylated, optionally N-protected4-amino-pyrimidinone, as shown in Scheme 2, followed by acylation andcyclisation. Alternatively 4 is first bicyclised and then condensed, asshown in Scheme 3.

Scheme 2 shows a Vorbruggen condensation (Chem. Ber. 1981, 114, 1234) ofthe differentially protected intermediate 4 with silylated4-amino-pyrimidin-2-one, wherein the 4-amino function is optionallyprotected with a convention amino protecting group as shown in Greene,“Protective Groups In Organic Synthesis,” (John Wiley & Sons, New York(1981)). Examples of such groups include: 1) acyl groups such as formyl,trifluoroacetyl, phthalyl, and p-toluenesulfonyl; 2) aromatic carbamategroups such as benzyloxycarbonyl (Cbz or Z) and substitutedbensyloxycarbonyls, and 9-fluorenylmethyloxycarbonyl (Fmoc); 3)aliphatic carbamate groups such as tertbutyloxycarbonyl (Boc),ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4)cyclic alkyl carbamate groups such as cyclopentyloxycarbonyl andadamantyloxycarbonyl; 5) alkyl groups such as triphenylmethyl andbenzyl; 6) trialkylsilyl such as trimethylsilyl; and 7) thiol containinggroups such as phenylthiocarbonyl and dithiasuccinoyl. The aminoprotecting group is of course selected such that its deprotectionconditions harmonise with the removal sequence of the differentialhydroxyl protecting groups. Alternatively Rs² is a synthon for theamides and imines defined for R⁵ and R⁶. In many cases no protectinggroup at all is required for the 4-amino function and thus Rs² is H.

As is conventional in Vorbrüggen condensation, the reaction mixturecontains TBDMSOTf and CH₂Cl₂ and the desired isomer 5 is separated withchromatography, for example HPLC. One of the hydroxyl protecting groupsin 5 is then selectively removed to uncover the hydroxyl function. Inscheme 2, compound 6, it is Rs which is removed first, and thedifferential pair of hydroxyl protecting groups can thus for example beTBDP (tert-butyl-silanyl) selectively removed with TBAF intetrahydrofuran for Rs, and MMTR (4-methoxy-phenyl-diphenylmethy)subsequently removed with acetic acid for Rs¹. However it is readilyapparent that other permutations of protecting groups will achieve thesame goal. Greene provides extensive reactivity charts over diverseprotecting groups to facilitate such selection. Additionally, swappingthe positions of Rs and Rs¹ by the appropriate manipulation of 4 willproduce an intermediate in which the 4-hydroxymethyl function isunmasked and acylated first.

The unmasked hydroxyl function in 6 is acylated with an activated,ωω-dicarboxylic acid HOOC-A-COOH, where A corresponds to —(CR¹R²)_(n)—as defined above to yield 7. In the event that R¹ or R² contain apotentially reactive group such as OH, NH₂ or COOH, these areconventionally protected as described in Greene ibid.

The activated acid used in the acylation may comprise e.g, the acidhalide, acid anhydride, activated acid ester or the acid in the presenceof coupling reagent, for example dicyclohexylcarbodiimide.Representative activated acid derivatives include the acid chloride,anhydrides derived from alkoxycarbonyl halides such asisobutyloxycarbonylchloride and the like, N-hydroxysuccinamide derivedesters, N-hydroxyphthalimide derived esters,N-hydroxy-5-norbornene-2,3-dicarboxamide derived esters,2,4,5-trichlorophenol derived esters and the like. Further activatedacids include those of the formula HCOOHACOOX where X for example isCOCH₃, COCH₂CH₃ or COCF₃ or benzotriazole.

Protecting group Rs¹ in Compound 7 is then removed to free up the 4hydroxymethyl group of 8 in preparation for cyclisation of the secondring of the bicyclic tetrahydrofuran ring system. This proceeds viaacylation as described in principle above.

Group Rs² of Compound 8, is then manipulated to produce the compounds offormula 1, as needed. For example an amino protecting group as Rs² canbe removed to yield the free amine at R⁵ & R⁶ and/or the amine functionconverted to an amide or imine as described below.

An alternative synthesis scheme for the compounds of formula I is shownin FIG. 3:

In scheme 3, the above-described differentially protectedtetrahydrofuran 4 is first bicyclised and then Vorbrüggen condensed.Bicyclisation proceeds via deprotection of a first of the Rs/Rs¹ pair ofcomplementary protection groups. In this case it is the 4-hydroxymethylfunction of the tetrahydrofuran which is first freed up ready foracylation, but this general methodology, with appropriate choice ofRs/Rs1 protecting groups can also proceed via removal and acylation ofthe 5 hydroxymethyl function as the first step to cyclisation.

The choice of 4- or 5-deprotection first is significant in those caseswhere A in the ωω-dicarboxylic acid HOOC-A-COOH is asymmetric, ie incompounds of formula I wherein m is 2 or 3 and wherein R¹/R² in thevarious methylene mers is not identical. For example where A is—CH(OH)CH₂— (that is in formula 1, n is 2, R¹ in the first methylenegroup is OH while R² is H, both R¹ and R² in the second methylene groupare H), then the localization of the R¹ hydroxy group adjacent the esterbond to the 4 hydroxymethyl function of the tetrahydrofuran intermediatecan be assured by deprotecting Rs first and using the activated acidPG-OC—CH₂—CH(OH)—COOH, where PG is a conventional carboxy-protectinggroup, which is of course selected such that its removal conditionsharmonise with the intended removal of RS¹. Greene provides extensivereactivity charts to facilitate such selection.

Carboxy protecting groups are extensively reviewed in Greene ibid andtypically comprise esters such as methyl or substituted methyl esters,for example methoxymethyl, methylthiomethyl, tetrohydropyranyl,tetrahydrofuranyl, methoxyethoxyethyl, benzyloxymethyl, phenacyl,including p-bromo, alpha methyl or p-methoxyphenacyl, diacylmethyl, orN-phthalimidomethyl. Ethyl esters include unsubstituted ethyl and2,2,2-trichloroethyl, 2-haloethyl, w-chloralkyl,2-(trimethylsilyl)ethyl, 2-methylthiethyl,2-(p-nitrophenylsulfenyl)ethyl, 2-(p-toluenesulfonyl)ethyl and1-methyl-1-phenethyl. Other esters include t-butyl, cyclopentyl,cyclohexyl, allyl, cinnamyl and phenyl, including m-methylthiophenyl.Benzyl esters include unsubstituted benzyl, triphenylmethyl,diphenylmethyl including bis(o-nitrophenyl)methyl, 9-anthrylmethyl,2-(9,10-dioxo)anthrylmethyl, dibenzosuberyl, 2,4,6-trimethylbenzyl,p-bromobenzyl, o-nitrobenzyl, p-nitrobenzyl, p-methoxybenzyl, piperonyl,4-picolyl and p-(P)-benzyl. Silyl esters include trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, i-propyldimethylsilyl andphenyldimethylsilyl. Activated esters include S-butyl, S-phenyl,S-2-pyridyl, N-hydroxypiperidinyl, N-hydroxysuccinimidoyl,N-hydroxyphthalimidoyl, and N-hydroxybenzotriazolyl. Miscellaneous estercarboxy protecting groups include O-acyl oximes,2,4-dinitrophenylsulfenyl, 2-alkyl-1,3-oxazolines,4-alkyl-5-oxox-1,3-oxazolidines and 5-alkyl-4-oxo-1,3-dioxolanes.Stannyl esters include diethylstannyl and tri-n-butylstannyl. Non-estercarboxy-protecting groups include amides such as N—N-dimethyl,pyrrolidinyl, piperidinyl, o-nitrophenyl, 7-nitroindolyl,8-nitrotetrahydroquinolyl and p-(P)benzenesulfonamide. Non-estercarboxy-protecting groups alos include hydrazides, such asN-phenylhydrazide or N,N′-diisopropylhydrazide.

An alternative scheme towards differentially protected tetrohydrofuranderivatives is shown in Scheme 4:

Scheme 4 is extensively reported in the academic literature. Thepreparation of the uridine analogue precursors is shown in Sanghvi et alSynthesis 1994, 1163, Sanghvi et al Tett Lett vol 35 p 4697 (1994) andHaly & Sanghvi Nucleosides & Nucleotides Vol 15 1383 (1996). Conversionof the uridine to cytosine analogues is shown in Kozlov, Nucleosides &Nucleotides vol 17 2249 (1998).

An alternative route to differentially protected tetrahydrofurans notrequiring conversion of the base i is shown in Scheme 5:

a: TrCl, pyridine, b: i) MsCl, Pyr ii) 1N NaOH, THF, c) i) ETAlCN, 65C,ii) toluene/THF, d) i) MsCl, Et₃N, ii) EtOAc, f) i) NaBH₄ ii) EtOH,α/β1:3-4, g) i) DIBAL, ii) silica gel EtOAc, epimerize α/β93:7, g) i)NaBH₄, ii) EtOH/CH₂Cl₂.

Although scheme 5 has been illustrated with a TrO protecting group andR⁵R⁶═H, it will be apparent that other variants for the amine andhydroxyl protecting groups will be amenable to this route.

Referring now to all schemes, imines where R⁵ and R⁶ together define an═CR⁸R^(8′) are typically prepared by condensation of the compound offormula I wherein R⁵ and R⁶ are, or the corresponding intermediate 5(optionally de-protected) with a compound of the formula(CH₃O)₂CHNR⁸R^(8′), typically in DMF at room temperature, analogously tothe procedure in Mauldon et al, Bioorg Med Chem 6 (1998) 577-585. Theappropriate formamide acetals are generally prepared from intermediatedialkylformamides and dimethyl sulphate at room temperature. Reaction ofthe intermediate salt with sodium methoxide provides semiacetals, whichare then condensed as described above.

Compounds of formula I wherein R⁵ is an amide are typically prepared byAkiyama acylation (Chem Pharm Bull 1978, 26, 981) of the N-4 unprotectedcompound of formula I, or the corresponding intermediate 5, with theappropriate ROOR in H₂O/1,4-dioxane. Compounds wherein R⁵ is an aminoacid residue are couple with an N-protected amino acid residue usingconventional peptide coupling conditions.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Various embodiments of the methods and compounds of the invention willnow be described by way of example only, with reference to the followingexamples and Figures; in which

FIG. 1 is a graph of the plasma concentrations over time of in-vivometabolite following oral administration of a compound of the inventionto rat;

FIG. 2 depicts inhibition of typical TAM strains having a primer rescuephenotype by the parent of the compounds of the invention relative toinhibition of conventional NRTIs, as further discussed in BiologicalExample 2a;

FIG. 3 depicts inhibition of M184V+TAMs having a primer rescue phenotypeby the parent of the compounds of the invention, relative to inhibitionof conventional NRTIs, as further discussed in Biological Example 2b;

FIG. 4 depicts inhibition of T69S+XX+TAMs by the parent of the compoundsof the invention, relative to inhibition of conventional NRTIs, asfurther discussed in Biological Example 2c;

FIG. 5 depicts inhibition of TAM strains by the parent of the compoundsof the invention, relative to inhibition by zidovudine and lamivudine,as further discussed in Biological Example 3

EXAMPLE 12-(4-amino-2-oxo-2H-pyrimidin-1-yl)-octahydro-1,5,10-trioxacyclopenta-cyclodecene-6,9-dione

aN-(1-{5-(tert-Butyl-diphenyl-silanyloxymethyl)-4-[(4-methoxy-phenyl)-diphenyl-methoxymethyl]-tetrahydro-furan-2-yl}-2-oxo-1,2-dihydro-pyrimidin-4-yl)-2,2-dimethyl-propionamide

To a solution of 0.75 g (1 mmol)4-amino-1-{5-(tert-butyl-silanyloxymethyl)-4-[(4-methoxyphenyl)-diphenyl-methoxymethyl]-tetrahydrofyran-2-yl}-1H-pyriminin-2-one,prepared as in Scheme 4 above, in dioxan (25 ml) under nitrogen wasadded a solution of di-tert-butyl dicarbonate (0.44 g, 2 mmol) in dioxan(2 ml). The reaction mixture was stirred at room temperature for 48 hrs.The reaction mixture was evaporated on silica gel and the residue waspurified on silica gel column using ethyl acetate/hexanes 2:1 as eluentto give 0.42 g (49%) of the product depicted above.

Proton NMR (CDCl3): 8.33 (d, 1H), 7.64-7.59 (m, 4H), 7.45-7.18 (m, 18H),6.91 (d, 1H), 6.79-6.77 (m, 2H), 6.10-6.08 (m, 1H), 4.08-4.06 (m, 1H),3.98-3.96 (m, 1H), 3.77 (s, 3H), 3.59 (dd, 1H), 3.19-3.16 (m, 1H),3.02-2.98 (m, 1H), 2.57-2.53 (m, 2H), 2.72-2.25 (m, 1H), 1.50 (s, 9H),1.08 (s, 9H).

b)(1-{5-hydroxymethyl-4-[(4-methoxyphenyl-diphenyl-methoxymethyl]-tetrahydrofuran-2-yl}-2-oxo-1,2-dihydro-pyrimidin-4-yl]-carbamicacid tert.-butyl ester

To a solution of the compound above (0.33 g, 0.4 mmol) intetrahydrofuran (10 ml) was added a solution of TBAF (0.19 g, 0.6 mmol)in tetrahydrofuran (1 ml). The reaction mixture was stirred at roomtemperature for 3 hrs. The reaction mixture was evaporated on silica geland the residue was purified on silica gel column using ethylacetate/hexanes 2:1 as the eluent. Evaporation of appropriate factionsgave 0.20 g (80%) of(1-{5-hydroxymethyl-4-[(4-methoxyphenyl-diphenyl-methoxymethyl]-tetrahydrofuran-2-yl}-2-oxo-1,2-dihydro-pyrimidin-4-yl]-carbamicacid tert.-butyl ester.

Proton NMR (CDCl3): 8.22 (d, 1H), 7.40-7.38 (m, 4H), 7.38-7.23 (m, 10H),6.85-6.82 (m, 1H), 6.03-6.00 (m, 1H), 4.04-3.94 (m, 2H), 3.85-3.81 (m,1H), 3.80 (s, 3H), 3.29 (dd, 1H), 3.11 (dd, 1H), 2.35-2.22 (m, 3H), 1.52(s, 9H).

c) Succinic acidmono-{5-(4-tert-butoxycarbonylamino-2-oxo-2H-pyrimidin-1-yl)-3-[(4-methoxy-phenyl)-diphenyl-methoxymethyl]-tetrahydro-furan-2-yl-methyl}ester

To a solution of the compound above (200 mg, 0.33 mmol) and4-dimethylaminopyridine (98 mg, 0.8 mmol) in dichloromethane (20 ml) wasadded succinic anhydride (80 mg, 0.8 mmol). The reaction mixture wasstirred at room temperature over night where after the reaction mixturewas added to a mixture of dichloromethane and sat. ammonium chloride.The organic phase was washed with water and dried. Evaporation of thesolvent gave 222 mg (94%) of the compound depicted above.

Proton NMR (CDCl3): 8.02 (d, 1H), 7.38-7.36 (m, 4H), 7.30-7.15 (m, 9H),6.84-6.81 (m, 2H), 5.89-5.87 (m, 1H), 4.58 (dd, 1H), 4.26 (dd, 1H),4.13-4.08 (m, 1H), 3.79 (s, 3H), 3.24 (dd, 1H), 3.05 (t, 1H), 2.80-2.60(m, 4H). 2.31-2.26 (m, 1H), 2.17-2.12 (m, 2H), 1.51 (s, 9H).

d) Succinic acidmono-[5-(4-tert-butoxycarbonylamino-2-oxo-2H-pyrimidin-1-yl)-3-hydroxymethyl-tetrahydro-furan-2-yl-methyl]ester

A solution of the compound above (222 mg, 0.31 mmol) in acetic acid (10ml) and water (5 ml) was stirred at room temperature for 3 hrs. LC/MSindicated complete conversion of the starting material to the desireddeprotected compound with a M+1 ion of 442. The reaction mixture wasevaporated to dryness and the residue was purified on a C-8 reversephase column eluted with acetonitrile/water 1:1.5 as eluent to give 100mg (73%) of the desired compound depicted above.

Proton NMR (CDCl3): 8.18 (d, 1H), 7.23 (d, 1H), 5.93 (broad s, 1H),4.66-4.63 (m; 1H), 4.33 (d, 1H), 4.15 (broad s, 1H), 3.66 (broad s, 2H),2.80-2.59 (m, 4H), 2.37 (broad s, 2H), 2.28-2.44 (m, 1H), 1.51 (s, 9H).

e)[1-(6,9-Dioxo-decahydro-1,5,10-trioxa-cyclopentacyclodecen-2-yl)-2-oxo-1,2-dihydro-pyrimidin-4-yl]-carbamicacid tert-butyl ester

To a solution of the compound above (74 mg, 0.168 mmol), HOBT (27 mg,0.2 mmol) and triethylamine (0.14 ml, 1 mmol) in dichloromethane (65 ml)and DMF (2 ml) was added EDAC (39 mg, 0.2 mmol). The reaction mixturewas stirred at room temperature for 48 hrs where after the reactionmixture was poured into dichloromethane (100 ml) and aq. Citric acid(100 ml). The organic phase was washed with sodium hydrogen carbonatesolution and brine. The organic phase was dried over sodium sulfate andevaporated to a residue which purified on a silica gel column usingethyl acetate as the eluent to give 22 mg (31%) of the compound shownabove.

Proton NMR (CDCl3): 7.75 (d, 1H), 7.36 (broad s, 1H), 7.25 (d, 1H),6.02-5.99 (m, 1H), 4.58 (dd, 1H), 4.36-4.28 (m, 2H), 4.14 (t, 2H), 2.64(s, 4H), 2.58-2.55 (m, 1H), 2.29-2.25 (m, 2H), 1.52 (s, 9H).

f)2-(4-Amino-2-oxo-2H-pyrimidin-1-yl)-octahydro-1,5,10-trioxa-cyclopentacyclodecene-6,9-dione

To a solution of the compound above (22 mg, 0.052 mmol) indichloromethane (2 ml) was added trifluoroacetic acid (2 ml). Thereaction mixture was stirred at room temperature for 1 h and evaporatedto dryness. Co-evaporation twice with toluene gave after careful drying12.7 mg of the captioned compound as the bis-trifluoracetate salt. LC/MSconfirmed the structure with characteristic ions of 324 (M+1) and 647(2M+1) and the HPLC purity was above 90% at 254 nm.

EXAMPLE 27-amino-2-(4-amino-2-oxo-2H-pyrimidin-1-yl)-octahydro-1,5,10-trioxacyclopentacyclodecene-6,9-dione

a) 2-tert-Butoxycarbonylamino-succinic acid4-{5-(4-tert-butoxycarbonylamino-2-oxo-2H-pyrimidin-1-yl]-3-[(4-methoxy-phenyl)-diphenyl-methoxymethyl]-tetrahydro-furan-2-yl-methyl}ester

To a solution of(1-{5-hydroxymethyl-4-[(4-methoxyphenyl-diphenyl-methoxymethyl]-4-tetrahydrofuran-2-yl}-2-oxo-1,2-dihydro-pyrimidin-4-yl]-carbamicacid tert.-butyl ester [98 mg, 0.4 mmol, described in Example 1] and4-methylaminopyridine (98 mg, 0.8 mmol) in dichloromethane (20 ml) wasadded N-Boc-(S)-asp anhydride [172 mg, 0.8 mmol (prepared as describedin J. Med. Chem. 1971, pp 24-30)]. The reaction mixture was stirred atroom temperature over night where after the reaction mixture was pouredinto ethyl acetate (150 ml) and sat. ammonium chloride (100 ml). Theorganic phase was washed with water, dried with sodium sulfate andevaporated to give a 371 mg of a crude product depicted above that wasused without any purification in the next step.

Proton NMR (CDCl3): 7.76 (d, 1H), 7.38-7.20 (m, 12H), 7.08 (d, 1H), 6.83(d, 2H), 6.13 (d, 1H), 5.80 (d, 1H), 4.83 (t, 1H), 4.61-4.58 (m, 1H),4.14-4.06 (m, 2H), 3.79 (s, 3H), 3.25-3.23 (m, 1H), 3.17-3.12 (m, 1H),3.00 (t, 1H), 2.80-2.76 (m, 1H), 2.26-2.15 (m, 3H), 1.56 (s, 9H), 1.4(s, 9H).

b) 2-tert-Butoxycarbonylamino-succinic acid4-[5-(4-tert-butoxycarbonylamino-2-oxo-2H-pyrimidin-1-yl)-3-hydroxymethyl-tetrahydro-furan-2-yl-methyl]ester

A solution of the compound above (330 mg, 0.40 mmol) in acetic acid (10ml) and water (5 ml) was stirred at room temperature over night. Thereaction mixture was evaporated to dryness and the residue was purifiedon a C-8 reverse phase column eluted with acetonitrile/water 1:1.5 aseluent to give 72 mg (32%) of the desired compound. LC/MS confirmed thecorrect structure with a molecular ion of 557 (M+1).

c)[1-(7-tert-butoxycarbonylamino-6,9-dioxo-decahydro-1,5,10-trioxa-cyclopentacyclodecen-2-yl)-2-oxo-1,2-dihydro-pyrimidin-4-yl}-carbamicacid tert-butyl ester

To a solution of the compound above (72 mg, 0.13 mmol), HOBT (20 mg,0.16 mmol) and triethylamine (0.07 ml, 0.5 mmol) in dichloromethane (50ml) and DMF (1 ml) was added EDAC (31 mg, 0.16 mmol). The reactionmixture was stirred at room temperature for 24 hrs where after thereaction mixture was poured into dichloromethane (100 ml) and theorganic phase was washed with citric acid solution, sodium hydrogencarbonate solution and brine. The organic phase was dried over sodiumsulfate and evaporated to a residue which purified on a silica gelcolumn using ethyl acetate as the eluent to give 26 mg (37%) of thecompound shown above. LC/MS gave the correct M+1 ion of 539 and M−1 ionof 537.

Proton NMR (CDCl3): 7.73 (d, 1H), 7.40 (broad s, 1H), 7.24 (d, 1H),6.02-6.00 (m, 1H), 5.26-5.24 (m, 1H), 4.87-4.85 (m, 1H), 4.64-4.55 (m,2H), 4.20-4.18 (m, 1H), 4.06 (t, 1H), 3.84 (t, 1H), 3.00-2.90 (m, 1H),2.76-2.66 (m, 1H), 2.57-2.52 (m, 1H), 2.31-2.17 (m, 2H), 1.52 (s, 9H),1.45 (s, 9H).

d)7-amino-2-(4-amino-2-oxo-2H-pyrimidin-1-yl)-octahydro-1,5,10-trioxacyclopentacyclodecene-6,9-dione

To a solution of the compound above (26 mg, 0.05 mmol) indichloromethane (2 ml) was added trifluoroacetic acid (2 ml). Thereaction mixture was stirred at room temperature for 2 h and evaporatedto dryness. Co-evaporation twice with toluene gave after careful drying24 mg of the title compound as the bis trifluoroacetate salt. LC/MSconfirmed the structure with characteristic ions of 339 (M+1), 677(2M+1) and 337 (M−1).

EXAMPLE 32-(4-Amino-2-oxo-2H-pyrimidin-1-yl)-octahydro-1,5,11-trioxacyclopentacycloundecene-6,10-dione

a) Hexanedioic acidmono-{5-[4-tert-butoxycarbonylamino-2-oxo-2H-pyrimidin-1-yl]-3-[(4-methoxy-phenyl)-diphenyl-methoxymethyl]-tetrahydro-furan-2-yl-methyl}ester

To a solution of(1-{5-hydroxymethyl-4-[(4-methoxyphenyl-diphenyl-methoxymethyl]-tetrahydrofuran-2-yl}-2-oxo-1,2-dihydro-pyrimidin-4-yl]-carbamicacid tert.-butyl ester [730 mg, 1.19 mmol, described in Example 1] and4-methylaminopyridine (350 mg, 2.86 mmol) in dichloromethane (80 ml) wasadded glutaric anhydride (327 mg, 2.86 mmol. The reaction mixture wasstirred at room temperature over night where after the reaction mixturewas poured into dichloromethane. The organic phase was washed withdiluted ammonium chloride solution, diluted citric acid solution, waterand brine and dried with sodium sulfate and evaporated to give 819 mg(95%) of a crude product depicted above that was used without anypurification in the next step.

b) Hexanedioic acidmono-[5-[4-tert-butoxycarbonylamino-2-oxo-2H-pyrimidin-1-yl]-3-hydroxymethyl-tetrahydro-furan-2-yl-methyl]ester

A solution of the compound above (1.09 g, 1.5 mmol) in acetic acid (50ml) and water (25 ml) was stirred at room temperature for 2.5 hrs. Thereaction mixture was evaporated to dryness and the residue was purifiedon a silica gel column eluted with EtOAc/MeOH 9:1 as eluent to give 435mg (64%) of the desired compound.

c)[1-(6,10-Dioxo-decahydro-1,5,11-trioxa-cyclopentacycloundecen-2-yl)-2-oxo-1,2-dihydro-pyrimidin-4-yl}-carbamicacid tert-butyl ester

To a solution of the compound above (395 mg, 0.87 mmol), HOBT (235 mg,1.74 mmol) and DMAP (213 mg, 1.74 mmol) in DMF (120 ml) was added EDAC(334 mg, 1.74 mmol). The reaction mixture was stirred at roomtemperature for 48 hrs where after the solvent was evaporated.Dichloromethane was added to the reaction residue and it was dilutedammonium chloride solution, diluted citric acid solution water andbrine, dried over sodium sulfate and evaporated to give 350 mg of acrude product. LC/MS showed that the desired product with ions at 438(M+1), 496 (M+acetate), 875 (2M+1) and 436 (M−1). Two purifications on aC-8 reverse phase column eluted with acetonitrile/water 1:1 andacetonitrile/water 1:1.25 gave, after evaporation and lyophilization, 31mg of the title compound with a purity of about 50% as determined byHPLC at 220 nM.

d)2-(4-amino-2-oxo-2H-pyrimidin-1-yl)-octahydro-1,5,11-trioxa-cyclopentacycloundecene-6,10-dione

To a solution at ±0° C. of the compound above (31 mg) in dichloromethane(2 ml) was added trifluoroacetic acid (2 ml). The reaction mixture wasstirred at ±0° C. for 2 h and then at room temperature for another 2 h.Thereafter the reaction mixture was evaporated to dryness and finallyco-evaporation with toluene gave a crude product that was purified onC-8 reverse phase column eluted with acetonitrile/water 1:2. Theappropriate fractions were evaporated after addition of TFA and 31 mg ofthe title compound as the trifluoroacetate salt was obtained. LC/MSconfirmed the structure with characteristic ions of 338 (M+1), 396(M+acetate) and 675 (2M+1) and the purity at 220 nM was about 70%.

Biology Example 1A Rat Pharmacokinetics

The compound of Example 1 was dissolved in MQ grade water, 3 mg/ml andorally administered to duplicate rats. The dose was 15 mg/kg and plasmasamples were taken at t0, 15 & 30 minutes, 1, 2, 4 and 6 hours. Recovery(as the metabolite2′,3′-dideoxy-3′-C-hydroxymethyl-β-D-erythropentofuranosylcytosine) inthe plasma was measured with mass spectrometry, detected as the sodiumadduct m/z 264 (M+Na)+.

As can be seen in FIG. 1, the compound of the invention provided asubstantial plasma concentration of the metabolite 2′,3′-dideoxy,3′-C-hydroxymethyl-β-D-erythropentofuranosylcytosine with a peakconcentration at this dose of around 4 uM. As rats cannot be infectedwith HIV, the antiretroviral activity of this formulation cannot bedirectly measured in this example, but it is noted that the ED₅₀ for themetabolite 2′,3′-dideoxy,3′-C-hydroxymethyl-β-D-erythropento-furanosylcytosine is typicallyaround 0.01 uM in human H9 cells. This in turn means that the peakplasma concentration is several hundredfold over the ED₅₀. Otherpharmaceutical parameters such as AUC and clearance are consistent withachieving a 24 hour trough level well over the ED₅₀ with QD or BIDdosing.

BIOLOGICAL EXAMPLE 1B Permeability

This example measures transport of inhibitors through the cells of thehuman gastroenteric canal. The assay uses the well known Caco-2 cellswith a passage number between 40 and 60.

Apical to basolateral transport

Generally every compound will be tested in 2-4 wells. The basolateraland the apical wells will contain 1.5 mL and 0.4 mL transport buffer(TB), respectively, and the standard concentration of the testedsubstances is 10 μM. Furthermore all test solutions and buffers willcontain 1% DMSO. Prior to the experiment the transport plates arepre-coated with culture medium containing 10% serum for 30 minutes toavoid nonspecific binding to plastic material. After 21 to 28 days inculture on filter supports the cells are ready for permeabilityexperiments.

Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denotedWash, row 2 “30 minutes” and row 3 “60 minutes”. Transport plate no 2comprises 3 rows of 4 wells, one denoted row 4 “90 minutes”, row 5 “120minutes and the remaining row unassigned.

The culture medium from the apical wells is removed and the inserts aretransferred to a wash row (No. 1) in a transport plate (plate no. 1) outof 2 plates without inserts, which have already been prepared with 1.5mL transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5. In A→Bscreening the TB in basolateral well also contains 1% Bovine SerumAlbumin.

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to theinserts and the cell monolayers equilibrated in the transport buffersystem for 30 minutes at 37° C. in a polymix shaker. After beingequilibrated to the buffer system the Transepithelial electricalresistance value (TEER) is measured in each well by an EVOM chop stickinstrument. The TEER values are usually between 400 to 1000Ω per well(depends on passage number used).

The transport buffer (TB, pH 6.5) is removed from the apical side andthe insert is transferred to the 30 minutes row (No. 2) and fresh 425 μLTB (pH 6.5), including the test substance is added to the apical (donor)well. The plates are incubated in a polymix shaker at 37° C. with a lowshaking velocity of approximately 150 to 300 rpm.

After 30 minutes incubation in row 2 the inserts will be moved to newpre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60minutes), 4 (90 minutes) and 5 (120 minutes).

25 μL samples will be taken from the apical solution after ˜2 minutesand at the end of the experiment. These samples represent donor samplesfrom the start and the end of the experiment.

300 μL will be taken from the basolateral (receiver) wells at eachscheduled time point and the post value of TEER is measured at the endthe experiment. To all collected samples acetonitrile will be added to afinal concentration of 50% in the samples. The collected samples will bestored at −20° C. until analysis by HPLC or LC-MS.

Basolateral to Apical Transport

Generally every compound will be tested in 2-4 wells. The basolateraland the apical wells will contain 1.55 mL and 0.4 mL TB, respectively,and the standard concentration of the tested substances is 10 μM.Furthermore all test solutions and buffers will contain 1% DMSO. Priorto the experiment the transport plates are precoated with culture mediumcontaining 10% serum for 30 minutes to avoid nonspecific binding toplastic material.

After 21 to 28 days in culture on filter supports the cells are readyfor permeability experiments. The culture medium from the apical wellsare removed and the inserts are transferred to a wash row (No. 1) in anew plate without inserts (Transport plate).

The transport plate comprises 3 rows of 4 wells. Row 1 is denoted “wash”and row 3 is the “experimental row”. The transport plate has previouslybeen prepared with 1.5 mL TB (pH 7.4) in wash row No. 1 and with 1.55 mLTB (pH 7.4), including the test substance, in experimental row No. 3(donor side).

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to theinserts in row No. 1 and the cell monolayers are equilibrated in thetransport buffer system for 30 minutes, 37° C. in a polymix shaker.After being equilibrated to the buffer system the TEER value is measuredin each well by an EVOM chop stick instrument.

The transport buffer (TB, pH 6.5) is removed from the apical side andthe insert is transferred to row 3 and 400 μL fresh TB, pH 6.5 is addedto the inserts. After 30 minutes 250 μL is withdrawn from the apical(receiver) well and replaced by fresh transport buffer. Thereafter 250μL samples will be withdrawn and replaced by fresh transport bufferevery 30 minutes until the end of the experiment at 120 minutes, andfinally a post value of TEER is measured at the end of the experiment. A25 μL samples will be taken from the basolateral (donor) compartmentafter ˜2 minutes and at the end of the experiment. These samplesrepresent donor samples from the start and the end of the experiment.

To all collected samples acetonitrile will be added to a finalconcentration of 50% in the samples. The collected samples will bestored at −20° C. until analysis by HPLC or LC-MS.

Calculation

Determination of the cumulative fraction absorbed, FA_(cum), versustime. FA_(cum) is calculated from:

${FA}_{cum} = {\sum\; \frac{C_{RI}}{C_{DI}}}$

Where C_(Ri) is the receiver concentration at the end of the interval iand C_(Di) is the donor concentration at the beginning of interval i. Alinear relationship should be obtained.

The determination of permeability coefficients (P_(app), cm/s) arecalculated from:

$P_{app} = \frac{( {k \cdot V_{R}} )}{( {A \cdot 60} )}$

where k is the transport rate (min⁻¹) defined as the slope obtained bylinear regression of cumulative fraction absorbed (FA_(cum)) as afunction of time (min), V_(R) is the volume in the receiver chamber(mL), and A is the area of the filter (cm²).

Reference Compounds

Category of absorption in man Markers % absorption in man PASSIVETRANSPORT Low (0-20%) Mannitol 16 Methotrexate 20 Moderate (21-75%)Acyclovir 30 High (76-100%) Propranolol 90 Caffeine 100 ACTIVE TRANSPORTAmino acid transporter L-Phenylalanine 100 ACTIVE EFFLUX PGP-MDR1Digoxin 30

Biology Example 2

Activity against TAM primer rescue-related resistant HIV in thePhenoSense HIV assay

The susceptibility of the compounds of the invention, measured as theplasma metabolite 2′,3′-dideoxy,3′-C-hydroxymethyl-β-D-erythropentofuranosylcytosine on HIV-1 isolatesfrom patient plasma samples that bear typical TAM primer rescue mutantresistant genotypes is determined by the commercially availablePhenoSense HIV assay (described in Petropoulos, C J et al., (2000)Antimicrob. Agents Chemother. 44:920-928 and performed by ViroLogics,Inc). The assay is performed by amplifying the protease (PR)—RT segmentof the HIV pol gene from patient plasma and inserting the amplificationproducts into a modified HIV-1 vector derived from an NL4-3 molecularclone.

Viral stocks are prepared by co-transfecting 293 cell cultures withrecombinant viral DNA vector and an expression vector that produces theamphotropic murine leukemia virus envelope proteins. Pseudotyped virusparticles are harvested from the transfected cell cultures and are usedto infect fresh 293 cell cultures. The recombinant viral DNA contains aluciferase gene cassette within the HIV env gene region and theproduction of luciferase in target cells is dependent on the completionof one round of virus replication. Drug susceptibility is measured byadding serial concentrations of the compound of the invention and thereference compounds to the cells. Drugs that inhibit virus replicationreduce luciferase signal in a dose-dependent manner, providing aquantitative measure of drug susceptibility.

EXAMPLE 2a

Table 1 summarizes a main cluster of primer-rescue-related TAM mutantsused in the experiment are resistant to HIV and bear the characteristicTAM genotype that typically emerges during AZT-involved antiretroviraltherapy.

Table 1. Characteristic genotype in primer rescue-related TAM patientisolates 20 and 21

Isolate number Characteristic primer rescue-related TAM mutations 20M41L, D67N, K70R, V118I, L210W, R211K, T215F, K219Q and L228H 21 M41L,D67N, K70S, V118I, L210W, R211K, T215Y, K219N and L228H

Results are depicted in FIG. 2. Wild-type HIV virus is used as thereference. Here, the inhibition of the patient isolate 20 and 21 strainsis expressed as the fold change in reduction of susceptibility to thetreatment drug as compared to parallel runs of the reference. Thefollowing antiviral drugs were tested: AZT, 3TC, TNF, ABC, d4T, FTC andthe compound of the invention, as the plasma metabolite 2′,3′-dideoxy,3′-C-hydroxymethyl-β-D-erythropentofuranosylcytosine. It is clearlyapparent that the compound of the invention retained activity againstthe TAM bearing strains. The results show only a 1.0 fold reduction insusceptibility for the isolate 20 strain and less than a 1.0 foldreduction in susceptibility for the isolate 21 strain. This means thatthe compounds of the invention retained activity against the patient'sprimer rescue-related mutant HIV RT at a level of potency similar to itspotency against wild type HIV RT. In contrast, other drugs, notably AZT(451 fold reduction in susceptability), but also to 3TC, TFN, ABC, d4Tand FTC, lost potency against the virus from these patients as comparedto wildtype. In other words, the virus from these patients exhibitedresistance, that is large reductions in susceptibility, to these drugsas shown in FIG. 2.

It is important to note that the two patient isolates harbor differentamino acid transitions at codon 215; T to F in isolate 20 and T to Y inisolate 21. This is a representative hallmark of primer rescue-relatedTAM resistance mutants.

EXAMPLE 2b

Table 2 outlines a primer rescue-related mutant HIV with the geneticbackground M184V (a discriminative mutant), which is typically selectedby the very commonly employed antiretroviral therapy AZT+3TC (Combivir).

TABLE 2 Genotypic changes in TAM- primer rescue-related patient isolate19 Isolate number Characteristic primer rescue-related TAM mutations 19M41L, D67N, K70R, V118I, M184V, L210W, T215F, K219E and L228H

As shown in FIG. 3, the compounds of the invention, as measured by theplasma metabolite 2′,3′-dideoxy,3′-C-hydroxymethyl-β-D-erythropentofuranosylcytosine once again retainedactivity against this resistant virus, showing only a 1.78-folddifference in susceptibility compared to wild type HIV. Both 3TC and AZTlost activity and showed reduced potency (i.e. a pronounced reduction inviral susceptibility) to the resistance virus (FIG. 3).

EXAMPLE 2c

Continuous challenge of patients with antiretroviral agents results inthe emergence of MDR. A T69S mutation with a 6-bp insertion betweenamino acids 68 and 70 in the finger region of RT is often seen incombination with various forms of TAMs and contributes to an enhancedprimer rescue activity. A cluster of MDR (with different forms of aminoacid insertion(s)) in combination with TAM was chosen, as outlined inTable 3.

TABLE 3 Genotypic changes in primer rescue-related patient isolates 31,32 and 35 Isolate Characteristic primer rescue-related TAM numbermutations 31 T69S + double amino acid insertion SG in the geneticbackground of TAMs A62V, D67E and R211K 32 T69S + double amino acidinsertion VG in the genetic background of TAMs A62V, D67G, V75I andT215I 35 T69S + double amino acid insertion VA in the genetic backgroundof TAMs A62V, R211K, T215Y and L228H

As shown in FIG. 4, the compound of the invention inhibited thesepatient isolates, giving the smallest change in drug susceptibilitycompared with six reference antivirals currently used in conventionalantiretroviral therapy.

Note that a pronounced (500 to 1000-fold) reduction in susceptibility toAZT was observed for patient isolates 32 and 35 whereas the compound ofthe invention showed changes of 2.79 and 4.29-fold respectively. This isconsistent with the compound of the invention displaying a differentmechanism of inhibition compared to the obligatory DNA chain terminatorsrepresented by conventional NRTIs.

EXAMPLE 2d

Isolate 4 represents a further discriminative mutant bearing theK65R+M184V genotype in a non-essential TAM background consisting ofmutations at R211S and K219E. This isolate causes a typicalcross-resistance to abacavir, 3TC and the newly approved nucleoside FTC,but retains its susceptibility to thymidine analogues, such as AZT andd4T. This isolate does not bear typical primer rescue mutations, yet thecompound of the invention still inhibits this viral phenotype asindicated by an FC value of 3.88. This value is comparable to thethymidine analogues, AZT (FC=1.11) and d4T (FC=0.71), whereassignificant resistance was found for 3TC (FC>200), FTC (FC>40) and tosome extent to ABC (FC>9.0). This experimental data demonstrates thatthe compound of the invention not only bears unique properties against“primer rescue” mutants but is also able to inhibit HIV mutants from thediscriminative family. This, therefore, contrasts with the inhibitorymechanism employed by 3TC and FTC as well as the likely mechanism of4′-C-ethynyl compounds in which M184V together with one additional aminoacid change in codon T165R in the catalytic region contributes tocross-resistance to 4-C-ethynyl nucleoside (Kodama 2002).

BIOLOGICAL EXAMPLE 3

Activity of 2′,3′-dideoxy-3-C-hydroxymethyl-cytosine against primerrescue-related resistant HIV in PBMC.

The antiviral performance of the compound of the invention againstadditional TAM primer rescue-related resistant HIV isolates was assayedin a PBMC culture. Isolates of HIV-1 were generated and expanded to hightiter by co-cultivation of infected patient PBMC with PHA-stimulateddonor PBMC (Virology Manual for ACTG HIV Laboratories). The cell-freesupernatants were harvested, sequenced, and stored in aliquots at −70°C. for drug susceptibility assays.

In vitro drug susceptibility assays were performed using a modifiedACTG/DOD consensus method (Virology Manual for ACTG HIV laboratories).PBMCs were pre-infected with viral stocks for 4 hrs at 37° C. in ahumidified atmosphere of 5% CO₂ following 4 hr incubation. Infectedcells were washed twice in media and pipetted into a microtiter platewith eight serial drug dilutions. Each well contained 100,000pre-infected PBMC and all drug dilutions were made with cell culturemedium. The drug dilutions were chosen to span the 50% inhibitoryconcentration (IC₅₀) for each single drug. Control wells containingcells and virus were co-incubated on each plate. After a 7-dayincubation at 37° C. in a mummified atmosphere of 5% CO₂, viral growthwas determined using a p24 antigen assay on supernatants (AbbottLaboratories, Chicago, USA). The percent inhibition of viral growthcompared to the control well, which contained no drug, was calculatedand expressed as fold changes (reductions in compounds susceptibility)compared to the control well. The reference compound AZT was run inparallel with the compound of the invention.

A cluster of representative of primer rescue-related mutant virus wasselected that harbors the essential feature of primer rescue-related TAMresistant RT mutations. Strains with mutations at position M41L, D67N,K70R, L210W, T215Y/F and K219Q/E in various combinations with or withoutdiscriminative mutant M184V were used as indicated in Table 4.

TABLE 4 TAM primer rescue-related genotype in 9 patient isolates Isolatenumber Characteristic primer rescue-related TAM mutations 1295 M41L,D67N, K70R, V75M, V118I, M184V, L210W, R211K, T215Y and K219E 7086 D67N,T69N, K70R, V118I, L210W, T215V and K219Q J12840 M41L, D67N, V118I,M184V, L210W, R211N, T215Y J10308 M41L, D67N, M184V, L210W, R211S, T215Y7141 M41L, D67N, M184V, H208Y, R211K, T215Y, K219N J14007 D67N, T69N,K70R, M184V, H208Y, R211K, T215F, K219Q, L228H VA206 D67N, M184V, L210W,R211K, T215Y VA286 M41L, E44D, D67N, L74V, V118I, M184I, E203K, H208Y,L210W, R211K, T215Y

Most of these selected primer rescue mutants conferred a pronouncedresistance to AZT susceptibility, dropping a couple of hundred folds inFC value. The exception was isolate 7086 (FC=3.0), which bears the T215Vamino acid mutation. A complete report of FC values is presented in FIG.5. Here, 2′,3′-dideoxy-3′-C-hydroxymethylcytosine inhibited all8-isolates, with the highest FC value being only 2.7.

All references referred to in this application, including patent andpatent applications, are incorporated herein by reference to the fullestextent possible.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

1. A compound of the formula I

wherein: R¹ is independently H, —OR³, —NHR⁴; C₁-C₄ alkyl; or, when n is2, adjacent R¹ together define an olefinic bond; R² is H; or when thegem R¹ is C₁-C₄ alkyl, that R² may also be C₁-C₄ alkyl; or when the gemR¹ is —OR³, that R² may also be —C(═O)OH or a pharmaceuticallyacceptable ester thereof; R³ is independently H, or a pharmaceuticallyacceptable ester thereof; R⁴ is independently H or a pharmaceuticallyacceptable amide thereof; R⁵ is H, —C(═O)R⁷, or an amide-bound L-aminoacid residue; R⁶ is H; or R⁵ and R⁶ together define the imine═CR⁸R^(8′); R⁷ is C₁-C₆ alkyl, C₀-C₃alkylcycyl; R⁸ and R^(8′) areindependently H, C₁-C₆ alkyl, C₀-C₃alkylcycyl; or R⁸ is H and R^(8′) is—NR⁹R^(9′); R⁹ and R^(9′) are independently H, C₁-C₆ alkyl,C₀-C₃alkylcycyl; or R⁹ and R^(9′) together with the N atom to which theyare attached define a saturated 5 or 6 membered ring; n is 1, 2 or 3;and pharmaceutically acceptable salts thereof.
 2. A compound accordingto claim 1, wherein R⁵ and R⁶ are H.
 3. A compound according to claim 1,wherein n is
 1. 4. A compound according to claim 3, wherein R¹ is H, OHor a pharmaceutically acceptable ester thereof.
 5. A compound accordingto claim 1, wherein n is
 2. 6. A compound according to claim 5, whereina first R¹ is H; and the second R¹ is —OH or —NH₂, or a pharmaceuticallyacceptable ester or amide thereof.
 7. A compound according to claim 6,with the formula:

wherein R⁵ and R⁶ are as defined in claim 1 and R₁* is said second R¹.8. A compound according to claim 5, denoted2-(4-amino-2-oxo-2H-pyrimidin-1-yl)-octahydro-1,5,10-trioxacyclopenta-cyclodecene-6,9-dione;or a pharmaceutically acceptable salt thereof.
 9. A compound accordingto claim 1, wherein n is
 3. 10. A compound according to claim 9, denoted2-(4-amino-2-oxo-2H-pyrimidin-1-yl)-octahydro-1,5,11-trioxa-cyclopentacycloundecene-6,10-dione,or a pharmaceutically acceptable salt thereof.
 11. A pharmaceuticalformulation comprising a compound according to any one of claims 1 to 10together with a pharmaceutically acceptable carrier or excipient.
 12. Acompound according to any one of claims 1 to 10 for use a medicament.13. A compound according to claim 12, for use in the treatment orprophylaxis of HIV.
 14. A compound according to claim 13, wherein theHIV is multiresistant HIV.
 15. A compound according to claim 14, whereinthe reverse transcriptase of the multiresistant HIV bears at least onemutation that allows an obligate chain terminating nucleoside- ornucleotide phosphate to be excised from the nascent DNA strand by ATP-or pyrophosphate-mediated excision.
 16. The compound according to claim15, wherein the reverse transcriptase bears at least one of thefollowing genotypic patterns: (a) M41, ±D67, L210 and T215; (b) D67, K70and K219; (c) T69S-XX; or (d) ▴67 (deletion at 67).
 17. A compoundaccording to claim 16 wherein the reverse transcriptase bears at least 3mutations.
 18. A method for the treatment or prevention of HIV infectioncomprising the administration of a safe and effective amount of acompound according to any one of claims 1 to 10 to a subject in needthereof.
 19. A method according to claim 18, wherein the HIV ismultiresistant HIV.
 20. Use of a compound according to any one of claims1 to 10 in the manufacture of a medicament for the treatment orprevention of HIV infection.
 21. Use according to claim 20, wherein theHIV is multiresistant HIV.